Curr Obstet Gynecol Rep (2012) 1:166–173

DOI 10.1007/s13669-012-0019-2

FEMALE INFERTILITY AND ASSISTED REPRODUCTIVE MEDICINE (Y ZHAO, SECTION EDITOR)

Recent Advances in Assisted Reproductive Technology

Published online: 6 July 2012

Abstract In 1978 the world witnessed the birth of the first infertile couples. As these relevant applications of ART
“test tube baby.” Since this time there have been explosive become increasingly utilized, it is incumbent upon society
advances in assisted reproductive technologies (ART). Cur- to ensure that these resources are made available in a mor-
rent optimizations surrounding the delivery of in vitro fer- ally responsible and equitable manner.
tilization (IVF) including the utilization of minimal
stimulation protocols and gonadotropin releasing hormone Keywords In vitro Fertilization . IVF . Preimplantation
(GnRH) agonist cycle triggers are being increasingly uti- genetic screening . PGS . Preimplantation genetic diagnosis .
lized to maximize patient safety. Modifications, such as PGD . Preimplantation genetic testing . Metabolomics .
those seen in the embryology laboratory, continue to im- Proteomics . In vitro maturation . Review . Embryo biopsy .
prove pregnancy rates. Concurrent with these advancements Fluorescence in situ hybridization . FISH . Embryo
in IVF have been the emergence of related technologies, cryopreservation . Oocyte cryopreservation . Egg bank .
such as embryonic genetic testing and oocyte preservation, Oocyte bank . Fertility preservation . Oncology . Time lapse .
which potentially have broad applications to both fertile and Videography . Fertility . Infertility . Infertile

Introduction
P. R. Brezina (*) : H. A. Zacur : T. A. Baramki : Y. Zhao
Department of Gynecology and Obstetrics,
In July of 1978, the world witnessed the birth of Louise
Division of Reproductive Endocrinology and Infertility,
Johns Hopkins Medical Institutions, Brown, the first successful in vitro fertilization (IVF) preg-
Phipps 264, 600 N. Wolfe Street, nancy achieved by Drs. Robert Edwards and Patrick Steptoe
Baltimore, MD 21287, USA [1, 2•]. Since that time, advances surrounding assisted re-
e-mail: [email protected]
productive technologies (ART) have experienced explosive
H. A. Zacur development and growth. Indeed, what was in 1978 per-
e-mail: [email protected]
ceived by many as a controversial medical curiosity has
Y. Zhao radically changed the prognosis of couples suffering with
e-mail: [email protected]
infertility and is responsible for an increasing portion of the
N. Ning world’s births [2•]. Many of the advances in ART have both
Department of Obstetrics and Gynecology, increased success rates and offered a broad range of options
The First Affiliated Hospital, Harbin Medical University, to couples undergoing treatment.
Harbin, China
Technological advances being developed and perfected
e-mail: [email protected]
currently hold the potential to change the field of ART in
E. Mitchell still more dramatic and exciting ways well into the future.
Department of Obstetrics and Gynecology, Some of these technologies, such as those focusing on the
University of Tennessee College of Medicine-Chattanooga,
979 East Third St., Suite C720,
genetic evaluation of developing embryos or oocyte cryo-
Chattanooga, TN 37403, USA preservation, impact the medical care of individuals both
e-mail: [email protected] with and without a diagnosis of infertility [3••]. In this
Curr Obstet Gynecol Rep (2012) 1:166–173 167

review we will discuss some of the cutting edge innovations incidence of IVF associated OHSS, significantly improving
in the field of ART and the implications of these technolo- the safety of women undergoing ART.
gies in the future. ART, as currently practiced, is also associated with an
increased rate of multiple gestation pregnancies [3••]. In IVF
cycles, this is principally a result of the routine transfer of
In Vitro Fertilization Advancements multiple embryos into the uterus [14]. Multiple gestation
pregnancies are clearly associated with increased health risks,
IVF has literally transformed the field of infertility since its both to mother and child, as well as increased medical costs
inception in 1978 [2•]. Initially, the techniques required to [14]. For these reasons, there is a concerted effort by many to
perform IVF were unrefined with relatively poor pregnancy limit the number of embryos transferred in IVF cycles. Many
rates [2•]. Major milestones in perfecting IVF, however, countries outside the United States have mandated that only
were developed quickly, including the use of controlled certain numbers of embryos may be legally transferred [3••,
ovarian hyperstimulation, luteal phase support, and im- 14]. The American Society for Reproductive Medicine
proved culture media [2•, 4, 5]. Other significant advances (ASRM) has made formal recommendations regarding this
soon followed with the development of intracytoplasmic issue (Pract Committee 2009 [15]). Many advocate the use of
sperm injection (ICSI), assisted hatching, and description single embryo transfer (SET) in IVF cycles, essentially elim-
of optimized ET techniques [6–8]. These advances both inating the risk of IVF associated multiple gestations. Several
improved the success rates associated with ART and in- clinics using SET report no decrease in the pregnancy success
creased the number of individuals that were candidates to rate while almost eliminating multiple gestation pregnancies
pursue infertility treatments [2•]. However, some trends in [16•]. The trend toward decreasing the number of transferred
the use of ART during this period were less than positive. embryos represents an improvement in ART that will signif-
Two of the chief concerns that emerged during the devel- icantly improve the cost and safety associated with IVF in the
opment of ART were the persistent risk of patients expe- coming years.
riencing ovarian hyperstimulation syndrome (OHSS) and/
or a marked increase in the risk of multiple gestation
pregnancies [3••]. Laboratory Advancements
The concept of controlled ovarian hyperstimulation
(COH) in the context of ART was pioneered by Howard Probably the single most significant factor in the dramatic
and Georgeanna Jones in the early 1980’s [9•]. This initial improvement in IVF pregnancy rates over the past 10-
COH protocol utilized 150 IU of human menopausal gonad- 15 years has been the technological modifications in the
otropin (hMG) which yielded an average of 3.7 oocytes embryology laboratory [2•]. Of these modifications, perfect-
retrieved per IVF cycles [10]. Later trials in the same center ing the embryo culture media has likely been the most
using higher doses of gonadotropins resulted in increased significant [2•]. Indeed, pregnancy rates increased dramati-
numbers of retrieved oocytes but did not improve pregnancy cally since the introduction of the culture media formula
rates [11]. In the late 1980’s and early 1990’s many centers titled “Human Tubal Fluid” first introduced in 1985 [17].
adopted increasingly aggressive COH dosing protocols Since that time, there have been essentially constant
which were associated with increases in various complica- improvements in the composition of embryo culture media
tions, the most notable being OHSS [9•]. that have continuously improved IVF outcomes [2•]. Vari-
Not only did these aggressive COH dosing protocols ous other modifications are constantly being evaluated to
result in increased risk, they also presented a significant optimize embryology laboratories. One such modification
financial burden on patients [9•]. Recently, there has been that is the subject of much research today is the optimal
an increased emphasis on exploring “minimal stimulation” oxygen (O2) concentration in embryo incubators [18]. Other
COH protocols [9•]. In several trials, utilization of such areas of ongoing research are evaluating the optimal pH of
protocols that utilize lower doses of injectable gonadotro- embryology media [19, 20]. In addition to media, various
pins results in comparable pregnancy rates with decreased other techniques have also been introduced that have opti-
medical complications and cost compared with more stan- mized outcomes. For example, assisted hatching and ICSI
dard COH protocols [9•, 12]. Another recent strategy for have been of significant benefit [2•, 7].
minimizing the risk of OHSS in COH cycles is the use of
gonadotropin releasing hormone (GnRH) agonist, instead of
human chorionic gonadotropin (hCG), to trigger ovulation Laboratory Advancements: In Vitro Maturation (IVM)
during an IVF cycle [13]. Taken together, minimal stimula-
tion COH protocols and the appropriate utilization of GnRH The ability to mature oocytes in a laboratory environment,
agonist to trigger ovulation have the potential to mitigate the in vitro maturation (IVM), is another technology that is
168 Curr Obstet Gynecol Rep (2012) 1:166–173

being currently perfected by many centers. IVM, as current- improvement in the protocols surrounding the processes
ly described, is the practice of retrieving immature human required for embryo cryopreservation and reanimation of
oocytes which then complete the transition from prophase I the embryo following the thaw [39].
to metaphase II, including extrusion of the 1st polar body, in A more recent advance in cryopreservation technology is
vitro [21–23]. Over the past decade or so, there have been the ability to cryopreserve unfertilized oocytes. Oocyte
many modifications in IVM protocols including priming with cryopreservation is a significant advancement in ART and
hCG, follicle stimulating hormone (FSH), and/or luteinizing has the potential to have a monumental impact on the field
hormone (LH) and specific changes in IMV oocyte culture of infertility as well as society in general. The first pregnan-
media [24–28]. Additionally, the retrieval procedures to obtain cy resulting from a cryopreserved oocyte was in 1986 [40].
oocytes for IVM are also continuously being perfected [29]. However, following this significant achievement, trials eval-
These modifications have resulted in several relatively small uating the pregnancy rates of cycles utilizing oocyte cryo-
trials that reported IVM pregnancy rates approaching, though preservation consistently yielded unacceptably low
still lower than, IVF cycles [30–34]. IVM does not appear to pregnancy rates [41]. These poor pregnancy rates were
introduce more risks, such as imprinting defects, as compared likely due to cellular damage during the cryopreservation
to IVF cycles [35, 36]. process [42•]. Early oocyte cryopreservation techniques re-
The chief benefit of IVM is often cited as a technology lied on controlled-rate or slow-cooling [41, 43•]. In 1999,
that could eliminate the risk of OHSS in PCOS patients [21, the first birth following oocyte cryopreservation using vitri-
22, 32]. However, the recent introduction of GnRH antago- fication was reported [44]. Since that time, oocyte cryopres-
nist cycles utilizing a GnRH agonist trigger and the in- ervation protocols using vitrification, including the
creased use of mild gonadotropin stimulation protocols introduction of the equipment innovations such as the cryo-
offer the ability to reduce or eliminate OHSS in otherwise top, have been continuously optimized [41, 42•, 43•]. These
conventional IVF cycles [9•, 12, 13]. Other investigators techniques resulted in oocyte survival rates following cryo-
have put forth IVM as a possible treatment for women who preservation approaching 90 % by 2005 [43•]. Currently,
have had suboptimal responses to traditional IVF cycles [31, multiple clinics report pregnancy rates in cycles utilizing
34]. The benefit of IVM in today’s current clinical environ- oocyte cryopreservation that approach those seen in fresh
ment is unclear and will need to be further explored. IVF cycles [45, 46]. However, the practice of oocyte cryo-
preservation is still considered an experimental technology
according to the American Society for Reproductive Medi-
Laboratory Advancements: Cryopreservation cine (ASRM)[47].
The demonstration that oocyte cryopreservation is now a
Perhaps the most significant advancement in embryologic viable alternative to embryo cryopreservation has broad
laboratory technology in recent years has been in the field of implications. The application of this technology that could
cryopreservation. The concept of gamete cryopreservation is impact the largest proportion of society is for women
not new. In 1942, cryopreservation and subsequent revival who wish to preserve their fertility. Women who pursue
of a mammalian sperm cell was first described [37]. Apply- professional careers are increasingly delaying childbear-
ing cryopreservation to IVF embryos was achieved relative- ing compared to historical norms[48]. While this trend
ly early in 1983 when the first pregnancy resulting from a has added to the richness and productivity of the world’s
cryopreserved embryo was reported [38]. The process used economy, it has also increased the rate of age related
to cryopreserve embryos has evolved since this time. For female subfertility and infertility [49]. While ART has
example, prior to cryopreservation today, embryos are ex- improved the prospects of achieving fertility for sub/
posed to low concentrations of glycerol and propanediol infertile women in their late 30’s and early 40’s, there
with sucrose supplementation to decrease the content of still exists a significant rate of failure to conceive for this
intracellular water [39]. These compounds result in decreas- population [49].
ing the intracellular ice crystal formation that may be asso- The introduction of the birth control pill in the latter half
ciated with cryopreservation [39]. The new technique of of the 20th century empowered women to have unprecedent-
vitrification requires decreased concentrations of these com- ed control to determine their desired involvement in the
pounds and therefore, in theory, may minimize theoretical workforce [50]. In a similar manner, the ability to preserve
concerns surrounding possible embryonic toxicity [39]. In- a woman’s fertility potential at a young age through oocyte
creased efficiency in performing embryo cryopreservation cryopreservation has monumental social implications. The
has increased ultimate pregnancy rates per stimulation cycle “biological clock” that women face as they approach their
and has provided increased flexibility within IVF cycles that late 30s and early 40s certainly influences career decisions
has proved critical for many functions including some forms and places disproportionate pressure on women, as com-
of preimplantation genetic testing [2•]. There is constant pared to men, to begin childbearing at relatively young ages
Curr Obstet Gynecol Rep (2012) 1:166–173 169

[49]. As oocyte cryopreservation technology continues to cells from the developing embryo and evaluating the genetic
improve and become less costly, its widespread use for composition of this cell(s) for either a specific genetic defect
elective fertility preservation by large segments of the pop- known to exist in the parents (PGD) or to screen for the
ulation seems likely. With this new opportunity, however, presence of embryo aneuploidy (PGS) [55•, 56]. The results
society must be mindful of economic barriers that would of this information then guide the decision as to which
likely lead to inequitable access to this technology that embryos are appropriate for embryo transfer [55•]. In
could further empower the options of the wealth at the 1990, Handyside et al. reported the first established preg-
expense of the poor. nancies using this procedure in two couples known to be at
Oocyte cryopreservation has additional applications that are risk for transmitting adrenoleukodystrophy and X-linked
currently being utilized. Oocyte cryopreservation is increas- mental retardation [57]. Since then, strides in molecular
ingly being offered as a method of fertility preservation for biology and IVF techniques have enabled the perfecting of
oncology patients prior to receiving chemo or radiation therapy PGD. PGD has successfully detected the presence of nu-
[51•]. The use of oocyte cryopreservation for the purpose of merous genetic based disorders, such as sickle cell anemia
fertility preservation is supported by professional societies and retinoblastoma [58, 59]. Advancements in genetic med-
[47]. Cryopreservation of unstimulated ovarian cortical tissue icine are increasingly linking medical conditions to specific
for the purpose of fertility preservation for oncology patients genetic markers [60]. The expansion of genetic medicine in
has also been performed, although with limited success [51•]. the future will certainly broaden the applications of medi-
At the present time, embryo and/or oocyte cryopreservation in cally indicated PGD.
the context of conventional IVF is the recommended method Another application for PGD is Human Leukocyte Anti-
of female oncologic fertility preservation. gen (HLA) typing [61]. This technology is generally
Another application for oocyte cryopreservation is the re- employed by parents who have a child affected by a partic-
cent creation of donor oocyte “egg banks” for use in donor ular disorder that could benefit from some sort of human
oocyte cycles. Traditionally, donor oocyte cycles demanded tissue transplant. For example, a child with leukemia who
that the cycles of the donor and recipient be coordinated [42•]. requires a bone marrow transplant. In these cases, PGD has
Egg banks eliminate the need for this time consuming and been employed as a modality to ensure that the next child
costly process while offering increased choices to couples that the couple conceives will be HLA compatible with their
undergoing a donor oocyte IVF cycle [42•]. As donor oocyte existing child with the given illness. This practice is rela-
cycles currently comprise approximately 10 % of all IVF tively uncommon but has generated considerable debate
cycles, egg banks, from a systems point of view, represent a regarding the ethics of HLA typing PGD [3••].
significant advance in quality and efficiency [42•, 52•]. In couples with recurrent pregnancy loss (RPL) and a
documented balanced reciprocal/Robertsonian translocation
or chromosomal inversion in one or both parents, preimpan-
Technologies Evaluating Embryos tation genetic diagnosis (PGD) coupled with IVF has been
shown to have some benefit in improving pregnancy and live
A significant challenge in ART has been and continues to be birth rates [62–64]. Traditionally, florescence in situ hybrid-
determining which embryos in a given IVF cycle are opti- ization (FISH) has been used to identify the presence of trans-
mal for uterine transfer. Traditionally, embryo morphology locations in PGD translocation cases. FISH is able to identify
has been the most utilized method of determining embryo both balanced and unbalanced chromosomal translocations. In
quality. There are numerous grading systems that have been recent years, microarrays are being increasingly utilized.
developed to grade embryo morphology [53, 54]. However, Microarrays are able to evaluate all 23 pairs of chromosomes
embryo morphology alone has been shown to be a subopti- and thus evaluate the chromosomes involved in the structural
mal indicator of determining which embryos have normal aberration as well other chromosomes for aneuploidy or other
chromosomal status (euploidy) or optimal implantation po- chromosomal imbalances [63, 65].
tential [53, 54]. For this reason, a variety of different mo-
dalities have been developed to evaluate embryo quality
both directly and indirectly. Technologies Evaluating Embryos: Preimplantation
Genetic Screening (PGS)

Technologies Evaluating Embryos: Preimplantation Chromosomal aneuploidy is believed to be the single great-
Genetic Diagnosis (PGD) est causal factor in pregnancy failure [66]. PGS is the
practice of evaluating cells from a developing embryo for
Preimplantation genetic diagnosis (PGD) and preimplanta- the purposes of identifying aneuploidy. PGS was introduced
tion genetic screening (PGS) involve obtaining one or more as a technology that could greatly improve pregnancy
170 Curr Obstet Gynecol Rep (2012) 1:166–173

efficiency in IVF patients at risk for miscarriage such as through PGS/PGD. This poses a risk for which couples need
couples suffering from recurrent pregnancy loss or patients to be appropriately counseled.
with advanced maternal age [67].
There are many different technologies that are used to
determine the ploidy status of embryonic cells for PGS. PGS Technologies Evaluating Embryos: Secretomics
for aneuploidy was first performed with FISH evaluation for and Metabolomics
approximately 5 chromosomes using a cell taken from the
embryo at the cleavage stage. FISH technology then pro- Secretomics and metabolomics offer another strategy of de-
gressed to the routine evaluation of 9-14 chromosomes termining which embryos are optimal to consider for uterine
[55•]. This PGS methodology resulted in suboptimal results transfer [75, 76]. These technologies attempt to determine
and was sharply criticized [68]. There are several reasons information about embryos from byproducts that can be mea-
why PGS using FISH at the cleavage stage did not produce sured in the media culture fluid surrounding the developing
optimal results. Firstly, FISH only evaluates a portion of embryo [75]. One important advantage of these approaches is
possible aneuploidies (9-14 out of 23 pairs of chromosomes that they provide a noninvasive manner to evaluate embryos
[56]. Therefore, FISH is unable to detect many chromosom- versus invasive techniques that require embryo biopsy.
al aneuploidies. Furthermore, high rates of aneuploidy/eu- Secretomics is the evaluation of specific protein profiles
ploid mosaicism are known to exist in cleavage stage found in the media culture fluid surrounding the developing
embryos [69]. Therefore, results obtained from a cleavage embryo [75]. The concept of secretomics was pioneered in
stage embryo may not represent the chromosomal status of animal models but has recently been applied to human
the remainder of cells comprising the embryo. embryos [77]. While a variety of methods have been used
In recent years, these two limitations have been addressed in the past, mass spectrometry using surface-enhanced laser
by the introduction of technologies that evaluate all 23 desorption/ionization coupled to time-of-flight analysis is
chromosome pairs and the ability to perform trophectoderm the most commonly utilized modality currently to perform
biopsy at day 5 of development (Fig. 1). The most common embryo secretomics [75, 78, 79]. Specific protein profiles
methods of performing 23 chromosome PGS employ micro- evaluated using this process has been shown by some inves-
array technology, utilizing either a single nucleotide poly- tigators to be predictive of implantation and pregnancy
morphism (SNP) or comparative genomic hybridization potential [78–80]. Some of these protocols focus on deter-
(CGH) platform [56]. Other forms of 23 chromosome PGS mining protein patterns that reflect cellular growth or apo-
evaluation include CGH on metaphase chromosomes and ptosis [80–82]. Other protocols are designed to detect
real time polymerase chain reaction (PCR) [70, 71]. PGS specific protein markers, such as ubiquitin, that are thought
evaluating 23 chromosomes and day 5 biopsy has resulted to be critical for embryo implantation [78, 79, 83]. Recently,
in excellent pregnancy rates in specific patient populations more invasive applications of this technology have been
such as couples suffering from recurrent pregnancy loss described that evaluate the fluid present in a blastocyst
(RPL) [72–74]. embryo [84]. However, this application of secretomics is
The recent technological advances in preimplantation experimental and not yet widely utilized.
genetic testing suggest that there will be a wider implemen- Metabolomics is the evaluation of the metabolic byprod-
tation of PGD/PGS in the future. However, PGD and PGS ucts present in embryo culture media [75]. These metabolic
require close collaboration between obstetricians, fertility byproducts contain complex metabolite patterns that may
specialists, IVF laboratory staff, and geneticists. Technical prove detailed information regarding the metabolic status of
limitations and the known phenomenon of mosaicism in the the embryo [75, 76]. The primary method of performing
embryonic complex can confuse diagnostic results obtained metabolomics is through the use of various spectroscopic

Fig. 1 These photographs show an embryo at the blastocyst stage. a shows the herniation of TE cells after the application of a laser to breach the
zona pellucida. b and c show the process of obtaining a sheet of TE cells that will be analyzed for PGS
Curr Obstet Gynecol Rep (2012) 1:166–173 171

techniques which are capable of identifying and comparing such as oocyte cryopreservation and preimplantation genetic
specific metabolites [75]. testing offer applications that may be applied socially on a
Metabolomics first developed by attempting to correlate grand scale. As these relevant applications of ART become
embryo potential to concentrations of specific metabolites. increasingly utilized, it is incumbent upon society to ensure
For example, early reports suggested that nitric oxide that these resources are made available in a morally respon-
metabolites may correlate with blastulation rates in devel- sible and equitable manner. This moral responsibility may
oping embryos [85]. Currently, however, the field is much prove to be one of the largest challenges surrounding ART
more complex and evaluates relative concentrations of nu- moving forward.
merous metabolites simultaneously [86•]. The specific
metabolites evaluated in these models are complex and
include carbohydrates, amino acids, carboxylic acids, fatty
acids, and nucleotides [86•]. Complicating matters further, Disclosure No potential conflicts of interest relevant to this article
there is a wide variation of “normal” for these compounds were reported.
and their relative concentrations change throughout embry-
onic development [86•]. Despite these challenges, however,
significant advancements utilizing metabolomics are cur- References
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