Indi an Journal of Experimental Biology
Vol. 39, January 2001, pp. 1-10
Review Article
Potential researchable areas in ARTs--Oocyte maturation
and embryo development
Tarala D Nandedkar & Radhika L Kelkar
Institute for Research in Reproduction, Mumbai 400 012, India.
In fertility is a co mmonl y encou ntered situation occurri ng equally in both sexes. In vitro fertilization and embryo transfer (IVF-ET) and other ass isted reproductive technologies (A RTs) have enhanced the possibilities for successful treatment to
tackl e in fert ility. However, ARTs currently face limitations due to the fact th at although success rate is high for the initial
stages such as ovulation induction and fertilization , it dwindles progressively so that the success rate of a take home baby is
as low as I 5-20%. Research centred around various stages in an IVF programme is therefore necessary to devise protocols
that ensure a hi gher success rate. This review takes a look at the potential areas currently under research in the field of
ARTs, such as, in vitro oocyte maturation, oocyte/embryo cryopreservation , embryo culture, preimplantation genetic diagnosis. Their applications, in clinical conditions such as cance r, have been di scussed.
Infertility is a problem that affects men and women ,
the causes and prevalence rate of which may vary.
The desire to reproduce is a highly motivating force
and failure to do so could prove to be a catastrophic
ex perience for the couple. Defined as a disease or
condition diagnosed after a couple has had 2 years of
unprotected coitus without conception; infertility is
quite common with one in every five couples being
affected at some point of time. While approximately
40% cases are due to the male factor and 40% due to
female factor, in the remainder 20% the causes may
be unexplained .
Under normal conditions, the course of events that
culminate into a pregnancy include-ovulation,
fertilization , cleavage and implantation (Fig. 1).
During each menstrual cycle, an oocyte from a
dominant follicle (selected from a pool of developing
follicles) is released into the fallopian tube. Following
ovul ation , fertilization occurs as a result of a single
sperm cell penetrating the barriers surrounding the
oocyte, thereby allowing fusion of male and female
pronuclei. The zygote so formed is transported from
the tube to the uterus during which it undergoes
multiple and rapid cleavages to form a mass of cells
called morula. In the uterus, the morula undergoes
further divi sions to form the blastocyst, so that, by
day five or six post-fertilization, the trophoblast cells
invade the uterine wall and implant the embryo for
further growth. Complex molecular interactions
directed by hormonal signals underlie the seemingly
simple processes mentioned above and any
di sturbances in the same could be a cause of infertility
in the female. Thus, female infertility may broadl y be
due to-{i) Blockage of fallopian tubes preventing
fertilization; (ii) Absence of ovulation ; (iii) Hormonal
deficiency ; and (iv) Non-receptive uterine lining.
Assisted reproductive technologies (ARTs) such as
in vitro fertilization and embryo transfer (IVF-ET),
gamete intrafallopian transfer (GIFT), zygote
intrafallopian transfer (ZIFf), intra-cytoplasmic
sperm injection (ICSI) can help the infertile women to
overcome the barriers to fertility. However, ARTs do
face certain limitations with age and ovarian
responsiveness, at play! . While fertilization rate is as
high as 80-90%, the success rate of a take-home baby
is 15-20%. It is therefore necessary to conduct
research in areas such as oocyte maturation, embryo
development and implantation to develop protocols
that ensure a higher success rate.
Fig. !- Diagrammatic represen tation of events durin g early pregnancy following ovulation : Release of the egg from the ovary;
fertili zati on and cleavage in the fallopian tube and transfer of the
embryo to the uterus and implantation .
2
INDIAN J EXP BIOL, JANUARY 2001
Oocyte maturation
Oocyte retrieval and culture-The first successful
human pregnancy from IVF-ET is to make use of a
single oocyte retrieved during a natural (nonstimulated) cycle. The time of aspiration is decided by
monitoring LH leve ls every 3 hours2 • However, the
low success rate associated with this approach led to
the research in 1980s that resulted in the development
of a number of stimulation treatment protocols. Use of
GnRH agonists to down-regulate the pituitary
followed by ovarian hyperstimulation using human
menopausal
gonadotrophin
(HMG)
and
superov ulation by HCG is a commonly used protocol
in IVF programmes. However, these stimulation
protoco ls have a number of disadvantages such as
high cost of hormones, multiple side-effects and a risk
of ovarian hyperstimulation (especially in case of
Polycystic Ovarian Syndrome patients).
In view of the above, development of alternati ve
treatment modalities for obtaining oocytes for IVF has
been a subject of intensive research in the past
decades. Two important strategies that have been
developed
and
being
further
investigated
include-{a) In vitro oocyte maturation (IVM) i.e.
retrieval of immature oocytes (1 00-120 J..lm diameter)
from antral follicles for maturation in vitro; and (b)ln
vitro follicle growth (IVG) i.e. growth of primordial
or preantral follicles to full size fo r subsequent IVM.
Immature human oocytes and intact follicles have
been obtained from ovarian tissue removed surgically
and cultured in vitro 3• Trounson et al. developed a
reliable in vivo method of transvaginal ultra-soundguided oocyte retrieval\ which has been further
refined by Russell et aZS. The requirements of
medium, nutritional factors, macromolecular and
hormonal supplements and culture conditions for
oocyte maturation have been reported by several
workers and presented in a number of recent
reviews 6-8 .
The first viable pregnancy using IVM oocytes was
reported by Cha et al. in 1991 followed by subsequent
reports 45 ·9• 10 • Similarly, attempts to grow primordial
follicles to Graafian stage have met with some
success as shown by studies of Eppig and O'Brien in
mice 11 , and Hovatta et al. 12' 13 and Oktay etal. in
humans 14 • However, a major problem that needs to be
tackled is the reduced developmental competence of
oocytes matured in vitro.
The process of oocyte maturation involves two
close ly integrated yet independent processes i.e.
nuclear maturation and cytoplasmic maturation.
nuclear maturation involves retntttatiOn of me iosis,
germinal vesicle breakdown (GVBD) and meiotic
progression from metaphase I to metaphase II. The
process of cytoplasmic maturation in vo lves changes,
such as, relocation of cytoplasmic organelles and
alterations in membrane transport systems that
prepare the oocyte for subsequent fet1ilization and
embryogenesis. Any imperfections in these processes
of oocyte maturation could subsequently compromi se
embryo viability.
An essential pre-requ isite to oocyte maturation is
the synthesis and storage of a vi de range of
molecules, both in the oocyte and cumulus cell s,
during oocyte growth. The mRNA and proteins
accumulated in the growth stage are necessary to
activate the maturation promoting factor which in turn
drives nuclear maturation 15 •
The molecular reprogramming that leads to oocyte
maturation is under the regulatory control of signals
produced by cumulus cells, the latter being produced
in response to gonadotrophin stimulation. Moor
et al. 16 thus suggest that the key to oocyte maturation
and embryo viability in vitro resides in the follicular
compartment rather than the oocyte. While Ca2+,
inositol-! , 4, 5-triphosphate, cyclic AMP, are known
to mediate cross-talk between oocyte and cumulus
cells during GVBD, factors that fine-tune meiotic
progression from metaphase I to metaphase II are
unclear. A number of candidate factors have been
suggested to play a role in follicular signalling that
directs oocyte maturation, such as, oestradiol 17B ,
EGF, activin, stem cell factor, thyroid hormone, Mos
protein (product of c-mos) matrix metalloproteinases
(Fig. 2) and have been extensively reviewed 15- 17 •
Oocyte diameter at the time of collection is another
factor that determines meiotic potential of oocytes in
vitro, as shown by Durinzi et al 18.
Follicular fluid steroids and proteins - markers of
oocyte quality--Steroid hormones present in the
ovarian follicular fluid are believed to play an
important role in oocyte maturation and acquisition of
developmental
competence of the
embryo 16 •
Consequently, estradiol levels are often monitored for
detecting matured follicles in gonadotrophinstimulated ovulation protocols. Besides being used as
a marker for ovulation induction, local estradiol levels
in the follicular fluid may also be used as a predictor
of pregnancy outcome 19 • Progesterone, an indicator of
ovulation and luteal function, can also be of a
prognostic value in pregnancy by IVP 0 . In a recent
study, a correlation between human chorionic
NANDEDKAR et al. : POTENTIAL RESEARCHABLE AREAS IN ARTS
gonadotrophin (HCG) in follicular fluid and granulosa
cells to oocyte maturity and fertilization has been
reported 2 1.
In addition to sex steroids, proteins and peptides in
the follicular fluid can help in determining the quality
of oocyte retrieval for ARTs and success of
pregnancy. The role of inhibins and activins in human
ovulation, conception and pregnancy have been
extensively studied and reviewed 22 . Many recent
studies have reported the potential use of these two
peptides as markers of ovarian reserve and oocyte
quality2 3-25 •
Oocyte preservation--During aspiration of oocytes
from ovaries of patients with stimulated cycles, as
many as 10 oocytes can be procured at a time. All
these may not be used for IVF and could be preserved
for revival and use in the next cycle.
Cryopreservation is a slow freezing method used
for preserving embryos/oocytes using various
cryoprotective agents (CPAs) such as ethylene glycol,
dimethyl sulphoxide (DMSO), 1, 2 - propanediol
(with or without sucrose) etc . These agents depress
the freezing point thereby reducing the extent of
intracellular ice formation and minimising the
extracellular salt concentration at a given
temperature 26 .
Despite the efforts of numerous workers, human
oocyte cryopreservation still remains a research
protocol with a success rate as low as 1% of living
babies per thawed oocyte27 . A number of reasons
could be attributed to the same, such as, low
fertilization due to a weakened zona, disorganisation
of the spindle apparatus and high incidence of
polyploidy 15 •28 ·29 . While zona drilling and ICSI are
3
being successfully used to overcome problems related
to the zona, solutions to prevent spindle
disorganisation, polyploidy etc. still remain elusive.
The process of vitrification may add a new
dimension to oocyte cryopreservation . While freezing
involves precipitation of water from the solution;
vitrification implies transition of an aqueous solution
as a whole from liquid to glass state (solid) bypassing
crystalline state26 • In a recent study by Isachenko and
Nayudu, the effect of temperature and egg yolk on
chromatin and spindle normality and cumulus
integrity was assessed, in mouse GV oocytes. Results
of the study ' have indicated that both pre- and postfreeze exposure of GV oocytes to 37°C with inclusion
of egg yolk has a positive effect on oocyte and
cumulus cell integrity with >80% normal survival
post thaw, 84% maturation rate and 45 % normal
spindle configuration 30 •
New approaches such as above may offer solutions
to some problems currently shadowing the field of
cryopreservation and in combination with IVM may
pave the way for the establishment of oocyte banks
facilitating oocyte donation for clinical cases of
premature menopause such as Turner's Syndrome 31 .
Another application of cryopreservation is in case
of cancer patients (Fig. 3). Aggressive chemotherapy
and/or
radiotherapy
for
malignancies
may
compromise ovarian function by depleting follicle
store thereby leading to infertility. In such cases,
cryopreservation of ovarian cortical tissue is one
strategy that holds promise for these patients in order
to restore fertility. A number of approaches for the
use of cryopreserved tissue are currently being
standardised since a substantial number of primordial
LH
-1-
--1-\-- >--Stem cell
factor
Activin
""
Activin
Fig. 2--Possible factors regulating oocyte maturation.
4
INDIAN J EXP BIOL, JANUARY 2001
follicles are found to be morphologically normal and
viable after freeze-thawing. Autografting the tissue at
an orthotopic site (laparoscopic insertion of cortical
biopsies into surgically resected ovary) or heterotopic
site with a rich vascular bed such as the kidney are
some of the options. These, however, may pose a risk
of reintroducing the malignant cells to the patient in
remission, especially in case of blood-borne or
ovarian cancers. Follicle isolation from thawed tissue
for in vitro growth and maturation is a possible
alternati ve, however, much remains to be standardised
in this approach 29 · 32 .
Embryo development
Embryo culture and transfer---ln human IVF-ET
progra mmes, intra-uterine embryo transfer is
routinely carried out on day 2 or 3 of development,
with an implantation rate of t5 %. An obvious
alternative for increas ing implantation rate would,
therefore, be the extended culture and transfer at
blastocyst stage. This will not only help in the
identification of those embryos with little or no
deve lopmental potential , but will also facilitate
synchronization of the embryo with the female tract.
Further, since the blastocysts have a higher
implantation rate than cleavage stage embryos, fewer
embryos can be used for transfer, reducing the risk of
multiple gestations which is a major problem
currently faced by IVF clinics24 •
Inspite of these proposed advantages, success rate
even in case of blastocyst transfer is lower, one
plausible reason being the failure to mimic adequately
in culture, the environment that the embryo
encounters in vivo. During its passage through the
oviduct and uterus, the embryo is exposed to dynamic
changes in the environment. Two approaches are
often resorted to for mimicking the same i.e. (i)
sequential changes in culture media, (ii) co-cultures
with epithelial cells.
Culture in sequential media--The concept of
sequential media is based on the dynamics of embryo
physiology and the everchanging requirements of the
embryo for two key nutrients. i.e. carbohydrates and
amino acids, wherein lies the need for the use of more
than a single culture medium. For instance, Gardener
and Lane have reported the use of two culture media
namely Growth 1 and Growth 2 (G 1 and G2) for
human embryo culture, up to blastocyst stage. While
G 1 medium is based on the carbohydrates and amino
acids present in the human fallopian tube at the time
of cleavage, G2 is based on conditions in the human
uterus
during
blastocyst
development
and
differentiation 33 .
An important component of all sequential media is
glycine which is catabolized into glycolate and
glyoxylate with release of ammonia. The latter, in
vitro, may form its carbonate or bicarbonate that are
highly unstable in alkaline pH. Glutamine is another
component of the medium and is needed before
genomic activation. Initially, glucose was used as the
main carbohydrate source in all medi a. Recentl y a
trend has emerged for reduction or complete omission
of the same from the culture medium wi th pyru vate as
the subsequent replacement 34 . However workers differ
in thei r opinion regarding the importance of glucose
in the medium 35 . Pyruvate has been advocated not
only to act as an energy source but also to detoxify the
ammonia in the embryo by transamination to form
alanine.
Equally important is the inclusion of albumin,
ethylene diamine tetraacetic acid (EDTA), insulin,
epidermal growth factor (EGF) in the medium. While
EDTA is added in the first step as the free radical
scavenger, it is omitted in the second step of culture
due to its effect on genomic activation. On the other
hand, growth factors are generally added in the
second culture medium 36 •
Co-cultures-Co-culture, as the term implies, is the
culture of the developing pre-embryo with epithelial
cells with a view to facilitate potential dynamic
interactions between the two. The cell . types
commonly used include tubal or endometrial
~
~
Ovarian ti ssue slices
(Cortical bio psy)
Cryopreservation
Thawing
Ma;.nual I Enzymatic
Grafting
isolation of follicles
Xenograft
Autogra ft
l
Orthotopic
site
Transplant
at hete.rotopic
stte
Natural
fertility
L
-01
SCID
1
.Q-·
l
Preantral follicles
~
~
~
Grow~
muturation
IVF-ET ~(--- of oocyte in -vitro
Fig. 3--Use of cryopreserved ovarian tissue to restore fertility in
cancer patients.
s
ANDEDKAR eta/. : POTENTIAL RESEARCHABLE AREAS IN ARTS
epithelium (of human or animal origin) autologous
human cumulus or granulosa cells or African green
monkey kidney cells (vera cell line). A number of
studi es evaluating the effect s of these cell types on the
developing embryo report an increase in the average
numbe r of blastomeres, decreased fragmentation and
he nce improved embryo development 37 ·38 . In one such
study , a significant improvement in the average
number of blastomeres per pre-embryo has been
reported, usin g an endometrial co-culture system set
up using one passage, freeze-thawed stromal and
glandular cells obta ined from the endometrial bi opsy
of the patient. However, the implantation and
pregnancy rate has been found to be the same between
the co-culture and non-co-culture cycles in patients
with repeated IVF failures 38 .
In contrast to thi s, some groups report no
difference in mea n cell number per pre-embryo and
blastulation rates usin g bovine oviductal or vero coculture systems 39 ..0 .Yet another effect of co-culture
seems to be improvement in the hatching rate and
implantation rate, where the former effect can be
attributed to the thinning of zona pellucida observed
during co-culture 4 1• On similar lines, co-cultures in
combination w ith assisted hatching seem to produce
beneficial effects in patients with poor prognos is or
repeated IVF failures , suggest ing that this approach
may hold a potential solution to the problem of
implantation failure due to impaired hatching 42· 43 .
An important issue to be taken into consideration is
the risk of bacterial or viral infection through the use
o f human hete rologo us cells or animal cells recovered
at the slaughterhouses or established cell lines. Use of
synthetic medi a that give a co-culture e ffect ha s been
suggested as an alternative in thi s case 4 1•
Factors regulating embryo development
Following fertilization , the egg unde rgoes a series
of cleavage di visions to form the blastocyst. However,
a significant number of embryos die during thi s
pre implantation peri od for reasons unexplained.
Several possibilities for the occurrence of the same
have
been explored, includi ng chromoso mal
abnormalities . genetic defects, unfavourable uterine
env ironment etc.
Aneuploidy, haploidy, polyplo idy, mosaicism are
some of the numeri cal chromosomal abnormalities
present in deve loping manJinalian e mbryos 44 • A study
by Viuff et a/. of bovine blastocysts developed in
vitro reports a high proportion of mixoploidy in these
blastocysts 45 • The significance of the phenomenon of
chromosomal abnormalities in prei mplantation human
embryos is reflected in a study wherein approximately
half of the spontaneous abortions in the first a nd
second trimester are chromosomally abnormal 46 •
During IVF, embryo quality is determined by two
main criteria name ly, the rate of development and
degree of fragmentation. The early stages of post fertilization development are controlled by maternal
factors stored in the egg. At some point during
deve lopment, the genetic information source is
switched to zygotic genes. Thi s switching of genetic
source or zygotic gene activation (ZGA) is reflected
in the alterations in protein expression pattern in
mouse oocytes and preimpl a ntation embryos 47 The
mouse Ped gene is a widely studied gene 111
preimplantation embryos. Localized to Q region of
the major histocompatibility complex (M HC). it
Antral follicle
Oocyte retrieval.
,!,
~Polar
bo: biopsy
PGD
IVF-ET
,!, 16- 18h
@
2-PN Oocyte
(Zygote)
In -vitro culture
Day 3
~ Post·insemm~tion
lffl'-x----+
Blastom<re biops~ 1 - ~ blastomorcs)
V;£0
PGO
6-10 cell stage
Day 5-6
~ Post-insemination
C \ - - +Biastocyst biopsy
. .
(upto 10 troplectoderm cells)
Blastocyst
Fig. 4---Methods to procure genetic material for PGD.
6
INDI AN J EX P BIO L. JANUA RY 200 1
encodes th e prote in Q a - 2 anti gen that dete rmin es the
rate of cleavage in the e mbryo. The e mbryonic Ped
gene is seen to be acti vated only after 2 - cell stage,
whi c h is the stage o f ZGA in mo use 48 • The search for
a human ho mo logue of the mouse Ped gene, however
is not co mplete.
A very co mmo n pheno me no n observed during
pre impl a ntati on deve lopme nt is the comple te or
parti al frag me ntati on of one or more blasto me res.
T he re is a gene ral consensus that degree of
fragmentati o n and deve lopmen ta l compe te nce a re
in verse ly related . Both necros is and apoptos is have
been suggested as possib le mec ha nisms res ul tin g in
fragme ntat ion. W hil e the fo rme r is a res ul t of a
physica l or c hem ica l inj ury, the latter is ge net ica ll y
reg ulated. Conseq ue ntly, stud ies have foc ussed on
two maj or gene fam ilies involved in apoptos is,
namely the Caspase and Bel - 2 fa mil y. Studi es to
assess mRNA leve ls for apoptotic genes re port
caspase mRNA in oocytes only while caspase
proteoly tic activity in polar bodi es a nd frag me nted
zygotes 49 O n the othe r ha nd , bax mRNA has been
found in all stages (incl ud ing oocyte) un like Bcl-2
mRNA wh ich has been detected onl y in zygote and
subseq ue nt p reimplantation stages 48 . Interesti ng ly,
Bcl-2 protein levels are lower in fragmented
blastocysts as compared to norma l ones suggesting a
role for Bc l-2 and bax in apoptotic e mbryos 49 .
However, in a stud y by A ntczak and B le rkom, no
correlation between apoptosis and fragme ntati on ca n
be establ ished. Instead a n assoc iat ion has bee n fo und
between patte rn s of frag me ntation a nd the parti al or
near-tota l loss of certain regul a tory protei ns th at occ ur
in polari zed domai ns in the mature oocyte and
e mbryo as we!J5°.
Assisted hatching-The tra nsfer of' the e mbryo to
the ute ru s is fo ll owed by impl a ntati on, whe re by the
blastocys t attaches itself to the uterin e wall for furthe r
An
esse nti al
pre-requi s ite
to
develop me nt.
implantation is the hatc hing of the blastocyst throug h
a series of contrac ti ons a nd expansions o f the
bl astocyst
a nd
rupture
of
zona
pe llucid a.
Conseq uently , impa irme nt of hatc hing has been
suggested as one of the reasons fo r a low impl antati on
rate. Ass isted hatch ing is ofte n resorted to in orde r to
overcome the sa me. A number of procedures are
e mployed for th is purpose , such as partia l zona
dissect ion (PZD) i.e. creating breaches in zona
mecha nic ally; zona drill ing usi ng acid Tyrode's
solution ; or employi ng a laser bea m w ith di rect
contact (UV) or without direct contact (IR). T hese
techniques have the ir ow n sha re of adva ntages a nd
di sad vantages, however, they ha ve been repo rted to
impro ve implantation rates 51.
Pre-implantation genetic diagnosis
Pre-impla ntation geneti c di agnos is (PGD ) as the
te rm suggests, is the geneti c analys is pri or to
impl antati on carri ed o ut to sc reen for e mbryos w ith
seri ous gene tic di seases 52 • It is an ea rl y form of
pre nata l di agnos is which he lps in the se lect ion of
un affec ted e mbryos for impl antati on o r at times, for
sex-based se lecti on of the e mbryo for transfer. PGD
offe rs the hi gh-ri sk coupl es the option of dec reas in g
the ri sk of tra nsmittin g inhe ritab le ge net ic d iseases to
the offsp rin g.
It has been nea rl y a decade since the first
pregna ncies were es tab lished after emp loy ing PGD to
sc reen for an X-linked defec t in couples at risk 5'.
S ince the n PGD has been used s ucces sfull y to sc reen
fo r s ing le ge ne defects of chromosomal abberrations .
In case of sing le gene defects with known genestruct ure, suc h as cystic fibros is o r Tay Sachs. D A
obtained from the e mbryo is analysed fo r the presence
of defect ive genes. On the other hand, if the gene
defec t is not known , a~ in case of Ducc hene M usc ular
Dys trophy (DMD) or he mop hili a. the sex of the
e mbryo is determined (s ince these defects occur in
ma les) a nd o nl y fema le e mbryos are tra nsfer red.
C hro moso mal abnormaliti es suc h as Down 's
Sy nd rome are sc ree ned by ka ryotyping i.e.
cytogenetic
a nalysis
of
ba nded
meta phase
c hromoso mes .
Sources of DNA for analysis- T he DNA sa mp le
needed fo r PGD may be obta ined (Fig. 4) e ithe r by
bi opsy o f (i) first po lar body fro m the unfe rtili zed
oocyte, or (ii ) one or more blasto me res from c leavage
stage e mbryo, or (iii ) trophectode rm ce ll s from the
bl astocys t.
(i) Po lar body bi opsy-T he fi rst po lar body for med
as a res ult o f the first me ioti c di vision of the oocyte
ca n be analysed w ithin 24 hr after the oocyte is
re tri eved . Thi s approac h is ofte n more eth icall y
acce pta bl e since the e mbryo is left unto uc hed.
However, the analys is in terms of the e mbryo is
indirec t since the polar body analysed w ill contain the
genotype of the oocy te on ly. Conseq ue ntly, the
method faces a drawback in that autosomal dominant
d isease in the male or sex determ ination cannot be
carried out.
(i i) B lastomere
biopsy-Here, one
or two
blastomeres are re moved fro m the e mbryo at the 6-8
NANDEDKAR et a/. : POTENTIAL RESEARCHABLE AREAS IN ARTS
cell stage (or day 3 post-fe rtilization ) and analysed .
Subsequently, embryos determined to be unaffected
are transferred to uterus at the end of day 3.
This is the most preferred approach since most
embryos attain this stage in vitro and since
blastomeres are totipotent, removal of one or two
blastomeres does not affect further embryo
development. This approach, however, falls short in
that only one or two blastomeres can be used, which
at times may not be sufficient 111 cases of
chromosomal mosaici sm44 .
(iii) Blastocyst biopsy-Co mpared to the above
two approaches, blastocyst biopsy offers much more
material for analysis, wherein upto ten trophectoderm
cells can be removed from the embryos cultured upto
day 5 or 6 post- fertilization. However, this approach
too has its shortcomings in that very few embryos
grow upto this stage in vitro and there is a possibility
that the biopsied trophectoderm cells may have
diverged genetically from the inner cell mass which
will later form the fetus.
Analytical techniques in PGD-The tools for PGD
may be divided into two categories based on direct
analysis of DNA , such as karyotyping, polymerase
chain reaction (PCR) and in situ hybridisation (ISH),
or an indirect analysis through gene products 54. The
latter for instance may involve biochemical assays to
enzyme
activity
during
various
monitor
developmental stages. However, no assays have been
standardised so far in human, probably due to the
activation of embryonic genome taking place
relatively late in development.
Karyotyping involves cytogenetic analysis of
metaphase
chromosomes
for
chromosomal
aberrations. The technique has its limitations due to
the time consumed and difficulty in obtaining cells in
metaphase. In this context, a recent study by
Willasden et al. may provide a rapid method for
karyotyping. The present study involved fusion of
human blastomeres with in vitro matured bov ine
oocytes with a view to force the blastomere nuclei
into metaphase using the metaphase inducing factors
present in unfertilized eggs. Of the 66 successfully
fused hybrid cells with human chromatin, 64(97 %)
yielded chromosomes that could be karyotyped .
Amongst the embryos from two patients with
maternal translocations, one embryo was found
normal and hence transferred leading to one
pregnancy with normal fetus. Studies such as these
may provide a basis for a rapid method for
7
karyotyping
blastomeres
from
preimpl antation
55
embryos .
The polymerase chain reaction (PCR) is usuall y
used for detecting single gene defects . Most PCR based techniques make use of nested PCR, wherein
two round s of PCR are carried out and the
oligonucleotide primers used in the second round are
specific for sequences internal to those sequences
recognised in the first round . This technique offers
several advantages such as increased sensitivity and
specificity and a reduced risk of carry-over
contamination. Fluorescent PCR, a modification of
PCR involving fluorescent nucleotide analogues
incorporated into the primers or amplified products,
helps in increasing sensitivity and accuracy of
detection . PCR - based techniques are be ing used
effectively for the detection of a number of single
gene defects causing diseases such as cystic fibrosi s,
Ducchene muscular dystrophy , Tay Sachs disease,
sickle-cell anaemia, B-thalassaemia, Lesch-Nyhan
syndrome, Huntington's disease etc.
Fluorescence in situ hybridisation ([F] ISH) is the
most reliable technique for detecting chromosomal
abnormalities, both numerical and structural , in preimplantation embryos, using fluorescent DNA probes.
The procedure involves spreading the single
embryonic nucleus on a slide with subsequent ISH
using fluorescent chromosome specific probes that
produce different colours for each chromosome
analysed. It is used widely for sex determination since
it can detect both X and Y chromosomes within a
single cell. Multicolour FISH (m-FISH) is a
modification of this technique using which, upto five
different chromosomes can be detected in a single
cell. Another modification known as primed in situ
labelling (PRINS) employs chromosome-specific
oligonucleotide primers for sex determination in case
of X-linked diseases.
Although applications of PGD are manifold in
ARTs, it raises a number of ethical, legal and moral
dilemmas52 . Despite these, there is a general
consensus among workers in this field that PGD is the
need of the day.
Embryo cryopreservation
During stimulation protocols, a considerable
number of mature oocytes can .be retrieved.
Consequently following subsequent fertilization and
embryo development, most IVF programmes resort to
embryo cryopreservation . The technique offers the
patients a solution for reducing the risk of multipl e
8
INDIAN J EXP BIOL, JANUARY 2001
pregnancies without destroyin g excess embryos which
can be used for subseque nt tra nsfe rs, if needed,
there by reducin g the need of ex pe nsive and time
consuming
gonadotrophic
stimulation.
The
procedures and cryoprotective agents employed a re
simil ar to th ose me ntioned prev iously for oocyte
preservati on, however, the tech nique re nde rs
sati sfac tory
results
in
case
of
e mbryo
c ryopreservati on even today , as compared to oocyte
freez ing 20 . Kaufman et al. have re po rted a promi sing
clinica l pregnancy rate of 21.7 % in a stud y in volving
1239 thawed blastocysts 56 . However, nega tive reports
do ex ist, the reasons for w hi c h could possibly be
attributed to two main factors - the process of
c ryopreservation
and
CPA
empl oyed.
Two
contrasting stud ies ca n be cited in thi s context. Park
et al have de monstrated hi ghe r e mbryo surviva l rates
in expand ed a nd ea rl y hatching bovine blastocysts by
vitrification usin g e lectron microscope grid s as
contai ners57 On the othe r hand , a comparison of the
effects of two c ryopreservation procedures name ly,
conve nti onal slow controlled rate freezing a nd
vi tri fica ti on was ca rri ed out by Uec hi eta!. Thi s stud y
has re ported a reduced functi o na l viabi lity in te rm s of
cr-I) 2-deoxyglucose uptake and bl astocyst
impl a ntati on rate in the vitrifi ed gro up 5R.
With the increas in g use of PGD, an important iss ue
to be conside red is whether the deve lopme ntal
potential of e mbryos is impa ired afte r manipul ati on
proced ures suc h as drilling or biopsy a nd
cryopreservatio n. So fa r, no human pregnancy has
been reported usi ng bi ops ied , c ryop reserved , thawed
e mbryos and lim ited data on thi s aspect is availabl e .
In a recent stud y by Jo ri s et al. , the effects of e mbryo
biopsy procedu re on survi va l after cryopreservation
has been assessed 59 • A signifi cantl y lowe r number of
bl asto me res is intact in the biopsied g roup as
co mpa red to the control group; and the survival rates
of biops ied e mbryos are red uced as well. However,
the surviving e mbryos developed to the blastocyst
stage ill vitro suggestin g a possibility of their
developme nt
to
term
by
dev ising
better
cyrop reservation protocols .
Alt hough w idel y e mployed , human e mbryo
cryopreservation raises many legal and ethical issues.
For insta nce, the creation of orphan e mbryos and thei r
disposal raises moral a nd eth ical dile mmas . In view of
these, oocyte cryopreservation seems to be a
preferable :dtern ati ve.
Despite
technological
advances
in
ARTs ,
invest igators in th is field face two maj or problems.
These
are--{i)
the
increasing
numbers
of
cryoprese rved embryos, and (ii) multipl e gestati on
rates. Although legislative actions have been taken in
some countries to tackle them, these problems
demand clinical and sc ie ntific soluti ons . The
incidence of multiple implantation brings with it a
physiological need for fetal reduction and an
increased risk of loss of pregnancy. For minimi si ng
this ri sk, it has been suggested that the numbe r of
embryos transferred should be restric ted to one or two
and the re mainder cryopreserved. Thi s howe ver,
would in turn contribute to the problem of
cryopreserved e mbryos. The need of the day , is
the refore, to ex plore other alternati ves by conductin g
research in areas such as ill vitro oocyte maturati on
and oocyte freezing. The story of A RTs would be a
true success story , if researche rs would deve lop
protoc o ls for successful s ing le e mbryo transfer,
e nabling IVF to trave l a full circle back to the birth of
Louise Brown. The first human IVF baby born as a
re sult of a s ing le e mbryo tran s fer.
Acknowledgement
The a uthors acknowledge the encourage me nt and
suggestions by Dr H S Junej a, Director, IRR .
Mumba i, India.
References
Spero ff L. Glass R H & Kase N G. Clinical gynecologic en docrin ology and infertility, 5th Edition (Wi lli ams & Wil kins )
1994. 932.
2 Steptoe P C & Edwards R C. Successful birth after lVF. Lan ·
eel , ii ( 1978) 366
3 Cha K Y, Koo J J. Choi D H. Hans S Y & Yoon T K. Pregnancy after in vitro fertil ization of human follicu lar oocytes
collected from non-stimul ated cycles, their cullllre in vitro
and their transfer in a donor oocyte program. Fertil Steril. 55
( 199 1) l 09.
4 Trounson A, Wood C & Kau sche A. In vitro maturation and
fe rtili zation and developmental competence of oocytes reco vered from untreated po lycystic ovarian patients. Fertil Steril.
62 ( 1994) 353.
5 Russell J B. Immature oocyte ret ri eval comb ined with in vit ro
oocyte maturation , Hu111an Rerrod. 13 (Suppl 3) ( 1998) 63 .
6 Leibfried - Ru tledge M L. Dominko T. Critser E S & Critser J
K, Tissue maturation in vivo and in vitro : Gamete and Early
Ontogeny, in Reproducti ve Tissue Banking - Scientific Prin ciples, edited by Karow A M and Critser J K, (Academic
Press. London) 1997. 23.
7 Salha 0 , Abusheika N & Sh arma V. Dynam ics of human fol licular growth and in vitro oocyte maturation. Hu111an Reprod
Update, 4 ( 1998) 816.
8 Smitz J & Cortvrindt R, Oocyte in vitro malllration and folli cle culture : Current dinical achievements and future directions, Jlulllan Reprod, 14 (Su ppl I) (1999 ) 145 .
NANDEDKAR eta/. :POTENTIAL RESEARCHABLE AREAS IN ARTS
9 Barnes F L. Kau sche A, Tigli as J, Wood C, Wilton L &
Trounson A, Production of embryos from in vitro matured
primary human oocytes, Fertil Ste ril. 65 ( 1996) 1151.
10 Jaroud i K A, Hollanders J M G, Sieck U V, Roca G L, El
Nour A M & Coskun S. Pregnancy after transfer of embryos
which were ge nerated from in vitro matured oocytes, Human
Rep rod, 12 ( 1997) 857
II Eppig J J & o· Bri en M J, Development in vitro of mouse oocytes from primordial follicles, Bioi Rep rod , 54 ( 1996) 197.
12 Hovatta 0 . Silye R. Abir R, Krausz T, Hardy K & Winston R
M L, Extracellul ar matrix improves survi val of both stored
and fresh human primordial and primary ovarian follicl es in
long term culture, Human Rep rod, 12 ( 1997) I032.
13 Hovatta 0. Wri gh t C. Krausz T, Hardy K & Winston R M L,
Human primordial , primary and secondary ovarian follicles in
long - tet m cu lture : Effect of partial isolation , !Iuman Reprod. 14 ( 1999) 2519.
14 Oktay R, Newton H, Mullan J & Gosden R G, Development
of human primordial follicl es to antral stages in SCID/hpg
mice sti mulated with follicle stimulating hormone, HwJW/1
Rep rod, 13 ( 1998) I 133.
15 Cha K Y & Chian R C. Maturation in vitro of immature human oocytcs fo r clinical use. /Iuman Reprod Updat e, 4
( 1998) 103.
16 Moor R M, Dai Y, Lee C & Fulka J Jr. . Oocyte maturation
and embryon ic failure, Human Rep rod Update , 4 ( 1998) 223.
17 Trounson A. Anderi esz C. Jones G M , Kausche A, Lolatgis
N & Wood C. Oocyte maturation. Human Reprod , 13 (Suppl
3) ( 1998) 52.
18 Durinzi K L. Saniga E M & Lanzendor f S E. The relationship
between size and maturation in vitro in th e unstimulated human oocyte. Ferri/ Steril, 63 ( 1995) 404.
19 Grego ry L. Ova ri an markers of implantati on potential in assisted reproducti on. Human Reprod, 13 (Suppl4) ( 1998) 117.
20 Nandedkar T D. Joshi N J. Shahid J K. Hinduja I, Correlation
of follicul~u· fl uid steroid concentrati on wi th concepti on in
patients undergoing in l'itro fertili zation , J Rep rod Bioi Camp
Endoc rin ol. 6 ( 1994) 19.
2 1 Enicn W M. Chantler E, Scif M W & El stein M, Human
ovarian granulosa cel ls and fo llicular fluid indices : The relatio nsh ip to oocyte maturity and fertili zation in vitro , Hun1an
Reprod, 13 ( 1998) 1303.
22 Lockwood G M, Muttukrishna S & Ledge r W L, lnhibins and
activi ns in human ovul ation , conception and pregnancy. 1/unwJI Rep rod Update, 4 ( 1998) 284.
23 Lau C P. Ledge r W L, Groome N P, Barlow D H & Muttuk ri shna S. Dimcric inhibi ns and acti vin A in human follicular fluid and oocyte-cumulus complex culture medium,
Human Reprod, 14 ( 1999) 2525.
24 Hall J E. Welt C K & Cramer D W. lnhibin A and inh ibin B
reflect ovarian func tion in assi sted reproduction but arc less
useful at predicting outcome . Huma11. Reprod, 14 ( 1999) 409.
25 Seifer DB , Lambert- Messerlian G, Hogan J W, Gardin er A
C. Blazar A S & Bcrk C A, Day 3 serum inhibin B is predicti ve of asststcd reproductive technologies outcome. Fo·ril
Steril. 67 ( 1997) II 0.
26 Kritscr J K. Agca Y & Gunascna K T. The cryobiology of
mammal ian oocytcs. in Reproducti i'C Tissue Banking - ScienriJic Principles . edited by Karow A M. Critser J K (Academic
Press, London) 1997, 345.
27 Mande lbaum J. Bela"isch - All art J, Ju nca A. Antoine J,
Plachot M. Alvarez S, AInot M. & Salat- Baroux J. Cryoprc-
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
9
servation in human assisted reproduction is now routine fo r
embryos but remains a research procedure for oocytes. Hu man Reprod, 13 (Suppl3) ( 1998) 161.
Saunders K M & Park J E, Effects of cryopreservation procedures on the cytology an d fertilization rate of in vitro matured
bovine oocytes, Bioi Rep rod , 61 ( 1999) 178.
Newton H. The cryopreservation of ovarian tissue as a strategy for preserving the fertilit y of cancer pati ents. !-Iuman Reprod Update , 4 ( 1998) 237.
lsachenko E F & Nayudu P L. Vi trifi cation of mouse germ inal vesicle oocytes : Effects of treatment temperature and egg
yo lk on chromat in and spindle normality and cumulus integrity, !-Iuman Reprod , 14 ( 1999) 400.
Foudila T, Soderstrom - Anttila V & Ho vatta 0 . Turner' s
syndrome and pregnanci es after oocyte donation , 1-fwnan Reprod, 14 (1999) 532.
Donnez J & Bassil S, Ind ications for cryopreservation of
ovarian tissue, !-Iuman Rep rod Update. 4 ( 1998) 248.
Gardner D K & Lane M, Culture of viable human blastocysts
in defin ed sequential serum - free medi a. !-Iuman Reprod, 13
(Suppl3) ( 1998) 148.
Coates A, Rutherford A J, Hunter H & Leese H J, Glucose free medium in human in vitro fertili zation and embryo tran sfer a large - scale, prospective, randomi zed clinical trial ,
Ferri/ Steril, 72 (1999 ) 229.
Bavi ster B D, Glucose and culture of human embryos, Ferri/
Steril, 72 ( 1999) 233.
Menezo Y, Veiga A & Bcnkhalifa M, Improved methods for
blastocyst formation and culture. Human Rep rod , 13 (Suppl
4) ( 1998) 256.
Vald M, Walker D & Kennedy R C. Nuclei number in human
embryos co-cu ltured with human ampullary cell s, Hum an Reprod, II ( 1996) 1678 .
Barmat L J. Liu H, Spandorfers S D, Kowalik A. Mete C. Xu
K, Veeck L, Damaria M & Rosewaks Z. Autologo us endometrial co - cultures in patients with repeated failures of implantation after in vitro fertili zation- embryo tran sfe r, J Assist
Reprod Genet, 16 ( 1999) 121.
Sakkas D, Jaquenoud N, Leppens G & Campana A, Comparison of results after in vitro fertilized human embryos are cultured in routine medium and in co-culture on vero cell s : a
randomized study, Ferr i/ Steril , 61 ( 1994) 52 1.
Tucker J M, Kort H L Toledo A A. Morton P C, Wright G,
lngargiola P E & Sweitze r C L, Effect of co-culture on subsequent survi val and implantation of cryoprcserved hum an embryos, J Assist Rep rod Gen et. 12 ( 1995) 689 .
Plachot M, Co-culture of embryos and fee der cel ls, Human
Rep rod, II (Suppl.l ) ( 1996) 35.
Weimer K E, Garrissi J, Steuerwald N. Al ikani M. Reing A
M, Ferrara T A, Noyes N & Cohen J, Bendicial aspects of
co-culture wi th assisted hatching whe n applied to multiple
failure in vitro fertili zati on pat ients, Hwna n Reprod, II
(1996) 2429 .
Weimer K E. Cohen J, Tucker M J & Godkc R A, The application of co-culture in assisted reproducti on : I0 years of experience with hu man embryos, Human Rep rod. 13 (Suppl 4)
( 1998) 226.
Mun ne S & Cohen J, Chromosome ab normalities in human
embryos, Human Rep rod Update , 4 ( 1998) 842 .
Viuff D. Rickards L, Offenberg H. Hyttel P. Avery B. Gre\'e
T, Ol saker I, Williams J L, Callcsen H & Thomsen P D. A
10
I DI AN J EXP BIOL, JANUA RY 200 1
high proportion of bovine blastocysts arc mixoploid. Bioi Reprod. 60 ( 1999) 1273.
46 Burgoyne P S. Holl and K & Stephens R, Incidence of numerical chromosome anomalies in human pregnancy estimation from induced and spontaneous abortion data, Hu111on Reprod. 6 ( 199 1) 555.
47 Sasaki R, Nakayama T & Kat o T, Microelectrophoretic
analysis of changes in protein expression pauerns in mou se
oocytcs and preimplantation embryos, Bioi Rep rod, 60 ( 1999)
1410.
48 Warner C M. Cao W. Exley G. McElhinny A S. Ali kani M.
Cohen J. Scott R T & Brenner C A, Genetic regulation of egg
and embryo survival. i-liullall Reprod, 13 (Suppl 3) ( 1998 )
178 .
49 Exley G E. Tang C. McElhinny A S & Warner C M, Expression of Caspase and BCL - 2 apoptotic family members in
mouse prei mplantation embryos. Bioi Rep rod, 61 ( 1999) 23 1.
50 An tczak M & Blerkom J V, Temporal and spatial aspects of
fragmentat ion in early human e mbryos : Possibl e effects on
developmental competence and association wi th the differential elimi nation or regu latory proteins from polarized domains, l-luiiiOII Reprod, 14 ( 1999) 429.
5 1 Mande lbaum J, The effects of assisted hatching on the
hatching process and implantation , llu111all Reprod, II (Suprl
I) ( 1996) 43.
52 Fasou liotis S J & Schenker 1 C, Preimpl antatio n ge neti c di agnos is principles and ethics, /Iuman Reprod, 13 ( 1998) 2238 .
53 Hand ysidc A H, Kontogianni E H. Hardy K &. Win ston R M.
Pregnancies from biopsied human preimplantation embryos
sexed by Y- specific DNA amplification . Naru re, 344 ( 1990)
768 .
54 Coonen E, Hopman A H N, Geraedts J P l'vl & Ramaekers F C
S, Application of in silll hybridization techniques to study
human preimplantati on embryos : A review, 1-lulllan Reprod
Update, 4 ( 1998) 135.
55 Willasden S, Levron J, Munne S, Schimmel T. Marquez C.
Scoll R & Cohen J. Rapid visualizati on of metaphase chromosomes in single human bl astomeres after fu sion with i11
vitro matured bovine eggs, 1-ftiiiWII Reprod, 14 ( 1999) 470.
56 Kaufman R A, Menezo Y, Hazout A, Nicollet B. DuM ont M
& Servy E J, Co-cu ltured blastocyst cryopreservation : Experience of more than 500 transfer cycles, Fertil Sterii , 64
( 1995) 11 25.
57 ParkS P, Ki m E Y, Kim D I. Park N H. Won Y S. Yoon S H.
Chung K S, Lim J H & Simpl e, efficient and successful vitrifi cation or bovine bl astocysts using electro n microscope
grids, Hu111an Rep rod, 14 ( 1999) 2838.
58 Uechi H, Tsutsumi 0, Morita Y, Takai Y & Taketani Y.
Comparison of the effects of controll ed - rate cryopreservation and vitri ficat ion on 2 - ce ll mouse embryos and subsequent development, 1-Iulltalt Reprod, 14 ( 1999) 2827.
59 Joris H, Van den Abbeel E, De Vos A & Van Steirteghem A,
Redu ced survival after human embryo biopsy and subsequent
cryo preservati on, Hwnwz Rep rod, 14 ( 1999) 2833.