Non-invasive oocyte quality assessment

Abstract

Oocyte quality is perhaps the most important limiting factor in female fertility; however, the current methods of determining oocyte competence are only marginally capable of predicting a successful pregnancy. We aim to review the predictive value of non-invasive techniques for the assessment of human oocytes and their related cells and biofluids that pertain to their developmental competence. Investigation of the proteome, transcriptome, and hormonal makeup of follicular fluid, as well as cumulus-oocyte complexes are currently underway; however, prospective randomized non-selection-controlled trials of the future are needed before determining their prognostic value. The biological significance of polar body morphology and genetics are still unknown and the subject of debate. The predictive utility of zygotic viscoelasticity for embryo development has been demonstrated, but similar studies performed on oocytes have yet to be conducted. Metabolic profiling of culture media using human oocytes are also limited and may require integration of automated, high-throughput targeted metabolomic assessments in real time with microfluidic platforms. Light exposure to oocytes can be detrimental to subsequent development and utilization of time-lapse imaging and morphometrics of oocytes is wanting. Polarized light, Raman microspectroscopy, and coherent anti-Stokes Raman scattering are a few novel imaging tools that may play a more important role in future oocyte assessment. Ultimately, the integration of chemistry, genomics, microfluidics, microscopy, physics, and other biomedical engineering technologies into the basic studies of oocyte biology, and in testing and perfecting practical solutions of oocyte evaluation, are the future for non-invasive assessment of oocytes.

Full text

Received: October 5, 2021. Revised: January 4, 2022. Accepted: January 11, 2022
© The Author(s) 2022. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please
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Biology of Reproduction, 2022, 1–17
https://doi.org/10.1093/biolre/ioac009
Oocyte Special Issue
Non-invasive oocyte quality assessment
Romualdo Sciorio1,Daniel Miranian2and Gary D. Smith2,3,*
1Edinburgh Assisted Conception Programme, EFREC, Royal Infirmary of Edinburgh, Edinburgh, UK
2Department of Obstetrics and Gynecology, University of Michigan, Ann Arbor, MI, USA
3Department of Physiology, Urology, and Reproductive Sciences Program, University of Michigan, Ann Arbor, MI, USA
*Correspondence: 4421 Medical Sciences I, 1301 E Catherine Street, Ann Arbor, MI 48109-5617, USA. Tel: +0017347644134; E-mail: smithgd@umich.edu
Abstract
Oocyte quality is perhaps the most important limiting factor in female fertility; however, the current methods of determining oocyte competence
are only marginally capable of predicting a successful pregnancy. We aim to review the predictive value of non-invasive techniques for the
assessment of human oocytes and their related cells and biofluids that pertain to their developmental competence. Investigation of the proteome,
transcriptome, and hormonal makeup of follicular fluid, as well as cumulus-oocyte complexes are currently underway; however, prospective
randomized non-selection-controlled trials of the future are needed before determining their prognostic value. The biological significance of
polar body morphology and genetics are still unknown and the subject of debate. The predictive utility of zygotic viscoelasticity for embryo
development has been demonstrated, but similar studies performed on oocytes have yet to be conducted. Metabolic profiling of culture media
using human oocytes are also limited and may require integration of automated, high-throughput targeted metabolomic assessments in real
time with microfluidic platforms. Light exposure to oocytes can be detrimental to subsequent development and utilization of time-lapse imaging
and morphometrics of oocytes is wanting. Polarized light, Raman microspectroscopy, and coherent anti-Stokes Raman scattering are a few novel
imaging tools that may play a more important role in future oocyte assessment. Ultimately, the integration of chemistry, genomics, microfluidics,
microscopy, physics, and other biomedical engineering technologies into the basic studies of oocyte biology, and in testing and perfecting practical
solutions of oocyte evaluation, are the future for non-invasive assessment of oocytes.
Summary Sentence
Direct and/or indirect non-invasive assessments of oocyte quality are reviewed and discussed in relation to clinical needs in assisted reproductive
technologies and as informative underpinnings of basic reproductive biology spanning follicle to embryo development.
Keywords: oocyte, oocyte maturation, chromatin content, non-invasive assessment, follicle, cumulus cells
Introduction
The oocyte morphological assessment represents a crucial step
in the assisted reproductive technologies (ART) laboratory
workday routine. Currently, it is generally very simple com-
pared to embryo and sperm assessment. Nonetheless, oocyte
quality is probably the most important limiting factor in
female fertility, playing a crucial role during the fertilization
process and subsequent embryo development [1]. According
to some authors [2], the phenotype of the adult stage offspring
is considerably correlated to the quality of the oocytes of ori-
gin. Indeed, the embryo is the result of the correct meeting of a
spermatozoon and an oocyte. The right selection of the oocyte
to be used for an in vitro fertilization (IVF) procedure would
be a logical tool for the reduction of the number of embryos to
be produced without changing the pregnancy outcomes. IVF is
a complex multistep procedure, the first of which is controlled
ovarian stimulation (COS). This process involves the use of
exogenous gonadotropins to stimulate the patient’s ovaries
to produce oocytes, which are then retrieved transvaginally
[3]. This application in ART further complicates the scenario.
Compared to the in vivo process, where oocyte maturation
occurs as the result of a long and natural follicle growth and
selection [4], common COS procedures suppress this natural
selection and allow successful maturation of oocytes that
otherwise would not develop spatially or temporally within
a pool of follicles. This may result in questionable and inher-
ently compromised quality, and could eventually be respon-
sible for fertilization failure, compromised embryo develop-
ment, or long-term consequences in offspring health [5].
It has been reported that even following intracytoplasmic
sperm injection (ICSI), human oocytes sometimes fail to fer-
tilize; those unfortunate events occur at an estimated rate of
3% [6]. Thus, this is a clear demonstration that successful
fertilization is a more complex process, related not only to
sperm penetration, but to many other processes inherent to
oocyte quality that are crucial and responsible for regulating
the majority of molecular and cellular mechanisms required
for successful fertilization. The concern is that a portion of
oocytes retrieved following COS is probably of compromised
quality; therefore, despite the optimal embryology condition
and efforts, they are eventually destined to fertilization failure,
or subpar embryo development, and/or the inability to estab-
lish a normal pregnancy. Furthermore, it is highly unlikely
that a single effector can account for these subpar outcomes.
The answer to these above queries, and many others in
reproductive biology, needs to be elucidated through a greater
understanding of regulatory mechanisms involved in oocyte
growth, maturation, and function [7].
We and others would propose that normal oocyte matu-
ration cannot be established simply by assessment of polar
body extrusion, but rather involves numerous cytoplasmic
processes not observed at the light microscope level. Those
mechanisms and signaling pathways in the oocyte cytoplasm
are vital for production and storage of carbohydrates and
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2Non-invasive oocyte quality assessment
proteins, successful position of organelles, and regulation
of metabolic pathways required for oocyte maturation and
competence to support normal fertilization and embryo devel-
opment. In other words, the oocyte quality likely relies not
only on the nuclear and mitochondrial genome, but on the
microenvironment provided by the ovary, mainly during fol-
liculogenesis, and the preovulatory follicle. This multitude of
effectors can modify oocyte transcription and oocyte transla-
tion, and therefore negatively affect the development potential
of the future embryo [8,9]. In this scenario, comparable to a
big puzzle, the artificial environment of the IVF laboratory
plays only a small role. The purpose of this review is to
evaluate and synthesize the predictive value of morphological
features of mature human oocytes—and their related cells
and biofluids—to their developmental competence. Since the
simple morphologic evaluation, before and after fertilization,
may not be the best measurement for oocyte competence, we
will discuss features of the oocyte, its surrounding environ-
ment, and potential analytical markers that may be required
to support more complete information about oocyte quality
and subsequent embryonic developmental competence.
The importance of oocyte quality in ARTs
Every oocyte likely has a different developmental potential,
and successfully predicting that potential is of great interest
for ART practitioners for a multitude of reasons. The
ability of an oocyte to undergo meiotic maturation has
been termed meiotic competence, and the ability to go on
to support successful embryonic development is defined as
developmental competence. In combination, these constitute
oocyte competence [10]. While maternal age is the best
predictor of oocyte competence [11], variation exists between
the competency of different oocytes retrieved during the same
COS, and currently, the most reliable methods of determining
oocyte competence are only marginally capable of predicting
a successful pregnancy [12]. Some of the most obvious
benefits of better predictive assessment of oocyte competence
are higher live pregnancy rates and fewer early pregnancy
losses [13]. An improved ability to predict oocyte potential
is also essential to the widespread adoption of elective single
embryo transfer (eSET), further reducing the morbidity and
higher costs of multiple gestations associated with multiple
embryos transferred [14]. Additionally, it is conceivable
that as the ability to predict oocyte competence continues
to improve, stimulation protocols could be optimized to
select for quality over quantity of oocytes retrieved, making
the experience more patient-friendly, with fewer cases of
ovarian hyperstimulation syndrome, and without sacrificing
outcomes [15]. The ability to forecast pregnancy outcomes
before oocytes are fertilized is also desirable, as some patients
are opposed to the creation of unused embryos and certain
countries have restrictive laws regarding embryo production
[13,16]. The growing cost associated with freezing additional
embryos could also be mitigated. Finally, improved non-
invasive assessment would allow better counseling to women
undergoing oocyte cryopreservation in the setting of fertility
preservation or for elective reasons [17].
Current oocyte nuclear maturation
assessment in relation to ARTs
In contemporary ART involving ICSI, the cumulus oocyte
complex (COC) must be denuded in order to remove all the
cells surrounding the oocyte, thus allowing visual assessment
of the sperm injecting procedure. The first step consists of
exposure to the hyaluronidase enzyme, followed by mechan-
ical force applied using pipettes of a diameter slightly larger
than the oocyte and surrounding cumulus. This allows accu-
rate assessment of oocyte nuclear maturation [GV-intact (GV-
I), metaphase I/anaphase I/telophase I (collectively termed MI)
or metaphase of meiosis II (MII)]. The assessment by light
microscopy can display the presence of the first polar body
(PBI) in the perivitelline space (PVS), which is considered to be
a marker of nuclear maturation. Oocytes with clear extrusion
of the PBI are supposedly at MII, with the chromatin aligned
on the equatorial plate of the meiosis II metaphase spindle.
However, recent studies using polarized light microscopy have
shown that a small percentage of oocytes displaying the PBI
in the PVS may still be immature at the telophase of meiosis I
stage [18]. In this case, a true oocyte maturation at MII might
be confirmed by the presence of the meiotic spindle (MS),
which can be localized underneath the PBI [19,20] and will
be discussed further below in relation to the use of polarized
microscopy. Additional studies have also examined the MS
and the orientation to the PBI to be used as a prognostic
tool. Oocytes with the MS separated >90◦from the PBI have
lower fertilization rates compared to oocytes where the MS
is oriented <90◦from the PBI [18]. However, a considerable
number of studies have analyzed the importance of the MS
orientation in human oocytes, and its presence has been
associated with fertilization rates and pregnancy outcomes
with generally contradictory results [21–27]. A contemporary
review on MS assembly, positioning, and function is avail-
able [28]. Nonetheless, it is important to consider that the
daily routine work in the embryology laboratory, including
manipulation and culture conditions, might have an effect
on the cytoskeleton of the oocyte, compromising the MS
and eventually being responsible for lower fertilization rates
[29]. However, following COS, it is generally expected that
around 80–85% of the oocytes are at the MII stage, with
clear extrusion of the PBI, whereas another 5–10% are at
GV-I stage and can be recognized by the spherical nucleus
containing a large exocentric nucleolus typical of the prophase
of meiosis I. Another 5–10% of oocytes with the absence of
both PBI and GV are classified as being at MI. These oocytes
have undergone GV breakdown, but have not fully pro-
gressed through meiosis I and are developmentally somewhere
between MI and MII, where the chromosomes are aligned on
the metaphase plate in preparation for finishing the first meio-
sis division [30]. The critical importance of proper chromatin
segregation/regulation during nuclear maturation and oocyte
cytoplasmic maturation in yielding oocytes with developmen-
tal competence has been reviewed elsewhere [31–33].
Defining non-invasive oocyte assessment and
its needs in ARTs
In IVF treatment, the selection of a single embryo to transfer
often presents a challenge to the embryology team, consider-
ing limited information available on an embryo’s viability. A
relevant percentage of embryos still fail to implant, despite
being morphologically scored as “top quality embryos,” or
even after having been genetically analyzed. As mentioned
earlier, the goal of an IVF cycle should be only one healthy
baby born per transfer; therefore, eSET should be regularly
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R. Sciorio et al. 3
employed in order to avoid the risk of prematurity and compli-
cations associated with multiple births [34]. The need to estab-
lish a non-invasive method to identify specific characteristics
of oocytes and embryos that reflect normal health function
or the ability to further develop into a healthy pregnancy is
wanting. To be non-invasive, a technology should have zero
detrimental impact on the oocyte, its fertilization, subsequent
embryo development, ability to establish a pregnancy, and a
healthy offspring with an expectation of a healthy lifespan
(similar to non-ART-conceived individuals). Going forward,
we will continue to discuss the current light microscope
non-invasive assessment of the oocyte. We will also consider
assessment of oocyte developmental competences that may be
invasive today, yet provide informative pathways forward that
may be non-invasive in the future. Finally, we will also discuss
non-invasive assessments that are making progress toward
utility in oocyte assessment.
The light microscopic appearance of the cytoplasm has been
the subject of several published studies that try to establish
its correlation with fertilization and pregnancy outcomes.
The definition of features differed between studies; there-
fore, relative analysis of results is difficult. Some authors
analyzed and defined the feature as “dark cytoplasm” [35,
36], whereas others used “diffused cytoplasmic granularity”
[37], “dark granular appearance of the cytoplasm” [38], or
“dark cytoplasm with slight granulation” [39]. When this
feature was analyzed and attempts were made to establish
a correlation with pregnancy outcomes, some authors found
that “dark cytoplasm” alone was not a predictive factor in
“in vitro” or “in vivo” parameters [35,38,39], whereas
others reported that embryo quality was compromised when
embryos developed from oocytes with dark cytoplasm [36,
40]. However, Wilding and colleagues [41] reported that cyto-
plasmic granulation was associated with higher fertilization
rates compared to oocytes with a total absence of granularity.
Therefore, the debate on dark cytoplasm and cytoplasmic
granulation is still alive, yet one can appreciate that these
observations are subjective and likely lack concordance within
and between laboratories.
Another recently investigated aspect is the relationship
between oocyte/embryo function and their biomechanical or
biophysical properties [42]. The viscosity of the cytoplasm
and the resistance of the cell membrane at ICSI were
analyzed by Ebner and collaborators [43] and Wilding
and coauthors [41]. Both viscosity and resistance had a
significant effect on outcomes such as fertilization, embryo
quality, and blastocyst formation. These cytoplasmic viscosity
differences have also been correlated with patients’ infertility
features and with ovarian stimulation regimens [43,44]. It
has been also reported that during follicle maturation and
COS, the oocyte cytoplasm viscosity might undergo several
changes—from an aqueous to a more viscous and sticky
substance. In the COS cycle that results in hyperstimulation,
the oocyte cytoplasmic maturation process often seems to
be out of sync when compared to nuclear maturation [43,
44]. In addition, viscosity in mature oocytes has also been
investigated, with findings revealing that higher viscosity
might be a poor prognostic indication of embryo development
and implantation [43]. Viscoelasticity in the cytoplasm of
mature oocytes and membrane resistance may be another
important feature to take into consideration, and perhaps
clarifying why some oocyte membranes break rapidly
at ICSI sperm injection, whereas those of other oocytes
fail to break even with aspiration [45]. Specific reports
have recently been published assessing viscoelasticity of
the oocyte and zygote as potential predictors of embryo
development [46] and will be discussed in further detail
below.
Non-invasive assessment of the follicular
environment and polar bodies
Analyzing follicular fluid (FF) is an appealing option for non-
invasive assessment of oocyte competence, as it is collected
during every oocyte retrial and is usually discarded. FF is
composed of granulosa and theca cell secretory products, as
well as the blood plasma components that cross the blood
follicle barrier [47]. Additionally, the FF is in intimate contact
with the maturing oocyte, and it is hypothesized that it may
contain evidence of either healthy or unhealthy developmental
conditions. Because the FF is likely different between oocytes
even during the same COS, one must be careful to only study
the associations between oocytes and their respective FF. This
creates some technical challenges, as increased needle sticks
during transvaginal oocyte pick up put the patient at increased
risk of bleeding, and the inherent dead space in the aspiration
tubing requires a certain amount of the buffer solution to be
wasted during each analysis. As a result, most FF studies have
looked at only the first oocyte retrieved from each ovary.
Follicular fluid transcriptomic as an indirect
non-invasive assessment of oocyte quality
Follicular fluid assessment has been categorized into the fol-
lowing groups for the purpose of this review: RNAs; pro-
teins (growth factors, anti-apoptotic factors, peptides, and
amino acids); hormones; reactive oxygen species; sugars; and
prostanoids. The term transcriptome indicates the total RNA
content inside a cell/biosample, including messenger RNA
(mRNAs), ribosomal RNAs, transfer RNAs, and microRNAs.
Analyzing the FF transcriptome is particularly challenging for
several reasons [48]. There are over 5000 genes known to be
expressed on the human oocyte [49] and the sheer number
of specific mRNA expressed by the oocytes may be likely
twice that number [50]. Additionally, because the FF also
contains mRNA from surrounding granulosa cells, providing
predictive information regarding oocyte potential from the
FF transcriptome using current techniques is limited. Since
mRNA is ultimately translated into proteins that have the
biggest impact on cell functionality and metabolism, analysis
of FF proteins (proteomics) or metabolites (metabolomics) has
proven to be more fruitful [48]. With this said, studies of
non-translated RNAs, such as long non-coding RNAs [51]are
beginning to be reported and may also have future predictive
value of oocyte developmental competence.
Follicular fluid proteomic as an indirect
non-invasive assessment of oocyte quality
One study analyzed the FF of healthy ovum donors using high-
pressure liquid chromatography (LC) at differing pH levels,
followed by mass spectrometry (MS) to separate a total of
742 proteins. This same study also used qualitative analysis to
compare samples of FF from aged matched controls of infertile
women undergoing IVF pre- and post-hCG administration,
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4Non-invasive oocyte quality assessment
and found 11 proteins that were significantly higher in the
pre-hCG samples and 6 proteins that were significantly lower
in the post-hCG samples. These proteins belonged to sev-
eral different functional groups, including protease inhibitors,
inflammation-mediated proteins, and cell adhesion proteins
[52]. Another study looked at the FF of three women with
recurrent pregnancy loss (RPL) and compared it to the FF
of three control women who had a history of at least two
prior births and no miscarriages. They found that the FF
of women with RPL had significantly less expression of
five different proteins, notably complement component C3c
chain E, fibrinogen γ, antithrombin, angiotensinogen, and
hemopexin precursor [53]. A different study looked at the
FF of women grouped as having either advanced maternal
age (AMA), a history of implantation failure, or AMA plus
a history of implantation failure, to see if there was an
association with polar body aneuploidy. They analyzed the
levels of FSH, LH, progesterone (P4), estradiol (E2), and Anti-
Müllerian hormone (AMH) and found that high FF FSH
levels were associated with PB aneuploidy. This may not be
surprising, however, there has been mixed evidence to date as
to whether the high doses of gonadotropins required for COS
lead to aneuploidy or not [54–61]. The other FF components
tested had no predictive value in aneuploidy, which is notable
because AMH FF has previously been shown to be a marker of
successful oocyte fertilization [62]. Because of the lack of clear
prognostic value when only one FF marker is used, some inves-
tigators have recently turned to analyzing a panel of markers
simultaneously. Recently, FF AMH and P4 levels of oocytes
that had been successfully fertilized using ICSI were both
analyzed together and grouped based on whether or not they
successfully progressed to the blastocyst stage. When looked
at in isolation, having a FF AMH level >15 pmol/L or a FF P4
level >60 mg/mL corresponded to a positive predictive value
of blastocyst formation of 76.96% and 90.99%, respectively.
However, when these same FF cutoffs were used in con-
junction, the positive predictive value of successful blastocyst
formation rose to 96.83% [63]. This was a relatively small
cohort of 88 patients and will need to be replicated, but it
again demonstrates the potential usefulness of FF proteomics.
Follicular fluid steroids and fatty acids as an
indirect non-invasive assessment of oocyte
quality
Steroids such as estrogen and testosterone in conjunction with
gonadotropins are believed to influence oocyte maturation,
but their exact role needs to be investigated further. It has been
shown that priming with estrogen is necessary for Ca2+oscil-
lations to occur during maturation, but improper estrogen-
to-testosterone ratios can lead to an abnormal Ca2+response,
thus negatively impacting embryo development [64]. Estrogen
(E2) and P4 within FF have been the target of investigation
for implications on pregnancy outcomes. It has been well-
documented that elevated FF E2 and an increased FF E2/P4
ratio are associated with a higher chance of achieving a
pregnancy [65–73]; however, there have been other stud-
ies that have not confirmed these findings [74–78]. Dehy-
droepiandrosterone (DHEA), is converted to testosterone by
theca cells in ovarian connective tissue [79]. The sulfatase
enzyme SULT2A1 reversibly catalyzes DHEA to its sulfate,
DHEA-S, which has been shown to be the precursor to
estrogen production in granulosa cells [80]. In fact, DHEA-
S is found in micromolar concentration in the FF, making
it the most abundant steroid in FF [81]. The interconver-
sion between DHEA and DHEA-S and their function in
both ovarian androgen and testosterone production has made
them logical targets for evaluation during IVF cycles. Interest-
ingly, initial studies looking at serum DHEA-S levels during
COS found no association with successful pregnancies [82],
whereas high DHEA levels in the FF were shown to have a neg-
ative correlation to oocyte retrieval number and subsequent
fertilization [83]. However, a more recent study looked at FF
DHEA-S and E2 levels of 521 women undergoing IVF. When
these women were stratified by low, medium, and high FF
DHEA-S, statistically higher pregnancy rates were seen in the
high FF DHEA-S cohort. There were also significantly more
total fertilization failures seen in the low FF DHEA-S cohort
compared to the medium and high FF DHEA-S groups. Raised
FF E2 levels were also seen in the high FF DHEA-S group,
as well as among the low and medium FF DHEA-S patients
who achieved pregnancy [84]. These findings corroborate the
role of estrogen in oocyte priming and suggest DHEA-S, as
a precursor to estrogen, may play an important role in this
mechanism.
Investigators have also looked to other steroid-related
molecules found in FF as potential prognostic markers. A
study conducted using the FF of the lead follicle of 54
women undergoing IVF found that oocytes that did not
show two pronuclei or underwent degeneration after ICSI
had significantly higher levels of arachidonic acid and linoleic
acid derivatives, as well as increased activity of secretory
phospholipase A. This study used strict selection criteria to
omit women over the age of 37, with BMIs ≥26, affected by
male factor infertility, or with poor IVF response [15]. These
fatty acids have been implicated in inflammatory activators
that may have an impact on the developing oocyte [85];
therefore, the selection criteria in the study was careful to
exclude conditions that may cause a baseline alteration in
inflammatory response or steroid levels. It is worth noting that
many of the studies excluded certain subsets of the infertile
population in their initial analysis, and larger studies including
more diverse patient populations will be needed to decipher
variations among different diseases and patient demographics
before these techniques can be reliably employed on a wide
scale.
Cumulus cells transcriptomics, metabolites,
and proteomics as indirect non-invasive
assessment of oocyte quality
Transcriptomic approaches can be used to explore the gene
expression level of cumulus and granulosa cells, in somatic
cells of the follicle, and in the embryos after fertilization [86].
The transcriptomics technology might help to determine new
diagnostic features indicative of oocyte and/or early embryo
development. Currently, assays of single gene activity can be
performed and associated with oocyte maturation, fertiliza-
tion, embryo competence, and future pregnancy outcomes.
Recent studies suggest that the individual transcript levels
of specific genes expressed in the COC are well-correlated
with oocyte maturation [87,88], embryo viability [87–90],
ongoing pregnancy [90–92], or live birth outcome [90,93].
However, it needs to be mentioned that the collection of cells
from the COC, although considered non-invasive, requires
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R. Sciorio et al. 5
the oocyte to be exposed to additional time outside the incu-
bator, which can be detrimental to subsequent development.
In addition, single COC culture needs to be performed in
order to retain the identity of each oocyte sample and its
resulting embryo, which can add extra cost and workload.
Nonetheless, the studies reported earlier are performed with
retrospective designs, without any clinical validation of the
identified markers. Further studies need to be completed with
the goal of identifying novel markers in the IVF setting that
will correlate to pregnancy outcomes [94].
The functional well-being of granulosa cells and/or cumulus
cells might be reflected in the oocyte quality [95,96]. During
the meiotic arrest, cyclic guanosine monophosphate from
cumulus cells goes into the oocyte through gap junctions and
impedes the hydrolysis of cyclic adenosine monophosphate
(cAMP) by the phosphodiesterase PDE3A. This inhibition
maintains a high concentration of cAMP in the oocyte and
keeps meiosis arrested. The preovulatory LH induces the
reduced levels of intra-oocyte cAMP and meiosis resumption
[97–99]. Growth differentiation factor 9 is a protein that, in
humans, is synthesized by the oocyte and plays an important
role in the expansion of cumulus cells, which is a critical aspect
of final follicle development [100,101]. Oocytes collected
from follicles with reduced expansion have a lower chance
of producing a good quality embryo and limited potential
for implantation (review: [102]). McReynolds and cowork-
ers [103] evaluated both the transcriptome and proteome
of human cumulus cells from young fertile oocyte donors
and infertile women of AMA and found striking differences
at the protein level. These investigators were able to iden-
tify 1423 cumulus cell proteins. When comparisons were
made between the abovementioned groups, statistical analysis
revealed 110 (7.7%) proteins to be differentially expressed.
Pathway annotation revealed significant involvement of pro-
teins in metabolism, oxidative phosphorylation, and post-
transcriptional mechanisms relative to AMA. In addition, gene
expression analysis showed altered profiles in cumulus cells
from women in their early to mid-40s. These types of analyses
are indicative of non-invasive assessment of oocyte-associated
transcripts and proteins that may have practical application in
the future. Prospective randomized non-selection-controlled
trials of the future will inform as to the reliability of these
cumulus transcripts and proteins as having a prognostic value
in determining oocyte developmental competence.
Polar body morphology and genetics as
indirect non-invasive assessments
of oocyte quality
The PBI is normally present in the PVS and is generally
smooth and without fragmentation. However, the impact and
the biological significance of PBI morphology, dysmorphisms,
or fragmentation are still unknown and a subject of debate.
Fragmented PBI should not be particularly considered an
oocyte marker, since the fragmentation may be related to the
post-ovulatory time. However, it has been suggested that a
degenerated PBI might be associated with asynchrony between
nuclear and cytoplasmic maturation, potentially related to
overmaturity of the oocytes [104]. Oocytes displaying a clear
and intact PBI have increased ability of producing blastocysts
and higher pregnancy rates [105,106]. Several studies have
been performed with the goal of determining the relationship
between PBI morphology and ICSI outcome, but were unable
to find a clear link between the two features [107,108].
Exceedingly large PBI seems to be a sign of poor prognosis
and is associated with compromised embryo viability [107,
109]. In addition, large PBI is related to high frequencies of
multinucleated blastomeres and might therefore contribute to
embryo aneuploidies [107].
It has been reported that the majority of aneuploidies in
human preimplantation embryos are derived from meiotic
errors occurring during oogenesis due to the lack of effi-
cient molecular mechanisms to repair eventual errors dur-
ing the meiotic division [110,111]. In addition, a growing
body of evidence suggests that environmental contaminants,
endocrine-disrupting chemicals, and air pollution, are posing
major threats to human reproductive health [112]. In that con-
text, preimplantation genetic testing for aneuploidies (PGT-A)
was introduced a few decades ago with the aim of identifying
euploid embryos to be transferred in IVF cycles [113]. Polar
body biopsy is an alternative technique to cleavage stage or
blastocyst biopsy first introduced by Verlinsky and colleagues
[114]. The main disadvantage of the PB biopsy technique is
that only the maternal aneuploidies can be identified; it will
not detect paternal meiotic or post-zygotic mitotic errors. A
considerable benefit of PB biopsy is the greater time available
to perform genetic testing without requiring embryo cryop-
reservation. An additional benefit is that PB biopsy avoids
embryo manipulation, which might be important in countries
where, due to ethical concerns, the manipulation of human
embryos is prohibited. In 2012, the European Society of
Human Reproduction and Embryology initiated a multina-
tional, multicenter, randomized clinical trial to assess the effec-
tiveness of PGT-A performed with PB biopsy by microarray
analysis. The main question of the study, named ESTEEM, was
to evaluate whether the analysis of 23 chromosomes in the PBI
and PBII and the selection of a euploid embryo for transfer
increased the chance of a live birth within one year among
women aged 36–40 years compared to ICSI without chro-
mosome analysis. Between June 2012 and December 2016,
205 consenting women were assigned to ICSI treatment with
PGT-A (study group) and 191 to ICSI without PGT-A (control
group) [115]. The results reported that the proportion of
women with a live birth within one cycle, followed by delivery,
was not significantly different between the two groups: 50
of the 205 with chromosome screening (24%) versus 45 of
191 without chromosome screening (24%). However, genetic
testing platforms are continually evolving, with microarray
analysis now being replaced by next-generation sequencing
(NGS), single-cell NGS, and non-invasive NGS.
Different methods have been proposed to identify aneuploi-
dies of all chromosomes in ARTs. Whole genome amplifica-
tion amplifies the genome and therefore results in an adequate
starting material for comprehensive chromosome screening
[116]. Other methods are metaphase comparative genomic
hybridization [117], array comparative genomic hybridization
[118], genome-wide single nucleotide polymorphism analysis
[119], PCR-based detection [120], and NGS [121], which is
becoming the procedure of choice for PGT-A, with improved
results compared to a-CGH [122]. In theory, although the
introduction of new molecular technologies should be able
to provide a better resolution for distinguishing aneuploidies,
there are some authors who have reported that those methods
may be intrinsically imperfect and require further optimiza-
tion [123]. Additionally, due to the small amount of DNA
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6Non-invasive oocyte quality assessment
collected from PBI, errors might occur more frequently during
the genetic analysis. Published studies reported a poor consis-
tency between the molecular analyses performed in different
laboratories, reflecting the potential of different molecular
technologies giving different interpretive information [124,
125]. It also must be noted that in most of these clinical PGT-
A analyses, the results are based on a single observation, an
n=1. This is concerning when one considers the standard of
care relating to clinical assays, be it chemistry, molecular biol-
ogy, microbiology, or genetics. Whether the genetic assessment
of the oocyte polar body with these newer platforms will yield
useful, practical, and informative selective power of oocyte
developmental competence is yet to be realized.
Non-invasive assessment of oocytes as a
direct evaluation of oocyte quality
Oocyte biomechanical properties
The study of biomechanical properties of cells and their result-
ing health, function, and development is becoming increas-
ingly appreciated. In both stem cells and ovarian cancer cells,
viscoelasticity has been shown to influence cell structure,
function, and biophysical properties [42,126]. Viscoelasticity
is defined as the property of materials that exhibit both vis-
cous and elastic characteristics when undergoing deformation.
Cell membranes are an example of a viscoelastic material
[127,128], and intracellular cytoskeletal organization and
remodeling have important relations to a cell’s viscoelastic
nature [129]. Studies have demonstrated that interactive bio-
physical forces, such as cell/membrane deformation and fluid-
flow shear stresses, impact cellular viscoelasticity [130–132].
It has been proposed that cell function and biomechanical
changes are a result of viscoelastic intracellular signaling
through kinase/phosphatase–structural cytoskeleton interac-
tions [133–136] and/or intracellular Ca2+[137]. While the
majority of these viscoelastic studies have been performed
in non-gamete cells, there are recent and significant reports
suggesting that measurements of oocyte biomechanical prop-
erties may provide insight into oocyte and subsequent embryo
function and development.
One oocyte-associated structure that may have significant
assessment discernment is the zona pellucida (ZP). The mam-
malian ZP is a sulfated glycoprotein matrix that is assembled
surrounding the primary oocyte during the early stages of
folliculogenesis [138]. The mouse ZP is composed of three
ZP glycoproteins, termed ZP1, ZP2, and ZP3 [139,140],
whereas the human ZP has four glycoproteins [141,142]. The
ZP serves as a molecular “gatekeeper” by providing species
specificity during binding of the spermatozoa to the oocyte
[143], stimulates the spermatozoa acrosome reaction [144],
and, following single-sperm binding and passage, is modified
to function as a component of the block to polyspermia [145,
146]. Using atomic force spectroscopy, Papi and colleagues
demonstrated that the ZP of immature eggs has a pure elas-
tic behavior, whereas in mature and fertilized oocytes, the
ZP transitions to a more plastic behavior—with a higher
force required to induce deformation [147]. It has been well-
recognized that following fertilization, the release of oocyte
cortical granule enzymatic contents into the PVS results in ZP
“hardening”—defined by both relative resistances to exoge-
nous proteolytic exposure (decreased solubility) [148] and/or
nanometrically measured mechanical properties [149]. Drob-
nis and coworkers performed early studies on direct measure-
ments of ZP “hardening” as a mechanical end-point measure.
These studies provided a much-needed understanding of the
relations of solubility and mechanical changes, as well as
a delineation of these two measures in relation to oocyte
maturity, fertilization, and species differences.
Independent of oocyte development, maturation, and/or
fertilization differences in ZP structure/function, the human
oocyte ZP’s association with embryonic developmental com-
petence and subsequent pregnancy success has also been inves-
tigated. Early works focused on ZP thickness measures and
ZP thickness variations across its two observed dimensions
[150,151]. Both of these studies reported that ZP thickness
variations of >20–25% were positively predictive of preg-
nancy success. In addition, birefringent microscopy, which
enables visualization of the inner, middle, and outer zones of
the ZP, has supported the idea that magnitude of light retar-
dance of the ZP inner layers may be a non-invasive measure
of oocyte developmental competence [44,152]. Collectively,
these retrospective, non-selective studies of ZP measures and
subsequent embryo development and/or pregnancy success
are encouraging as potential non-invasive oocyte assessments;
however, properly powered prospective randomized trials are
wanting.
Elegant studies performed by Yanez and colleagues have
demonstrated the ability to measure viscoelastic properties
on mouse and human zygotes [46] using a micropipette aspi-
ration platform that allowed quantification of the depth of
aspiration of the ZP, and part of the cell membrane, over
time. These investigators reported the ability to quantify four
biomechanical parameters of a linear elastic solid model for
each zygote measured. While no individual parameter had pre-
dictive value in relation to subsequent embryo development,
combining parameters showed partitioning of zygotes des-
tined for blastocyst development [17,46]. While these studies
are promising and thought-provoking, such studies remain to
be performed on oocytes before fertilization. Additionally, as
mentioned above, prospective randomized controlled human
studies are needed to decipher if these viscoelastic measures
are predictive of developmental competence and pregnancy
success, and if they are accurately defined as non-invasive.
With that said, these and other performed and proposed
studies that integrate biomechanics, single-cell analysis, novel
means of microscopy (see: following sections), microfluidics,
and oocyte developmental biology likely will have research
and practical utility as non-invasive means of assessing and
quantifying oocyte developmental competence (review see:
[17,153,154]).
Oocyte metabolomics
Metabolomics is classically defined as the non-targeted
identification—and, importantly, quantification—of all low
molecular weight end products of metabolism (metabo-
lites) [155]. As the number of metabolites are reduced
approximately, two magnitudes reduced in comparison to
genomic and transcriptomic measures, it has been proposed
that metabolomic analysis can be performed more rapidly
[48]. The goals of metabolomics are to quantify either
individualized metabolites as biomarkers, or metabolic
fingerprints (grouping of multiple metabolites), in a cell
system under comparative physiological conditions at a given
developmental temporal set-point [156]. Two important
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R. Sciorio et al. 7
concepts make metabolomics attractive as a non-invasive
assessment of cellular function. First, it has been proposed
that cellular health can be delineated by the modified
concentrations of metabolites reflective of attempts to
maintain metabolic homeostasis [157]. Second, the products
of intracellular metabolism—the resulting metabolites—are
in dynamic balance with those in biofluids surrounding
the cells, whether those fluids represent plasma, FF, or
culture media [155]. For these reasons, and others men-
tioned below, the presumption that metabolic profiling of
individual or groupings of metabolites in culture media
of oocytes and/or embryos in ART might provide non-
invasive measures of cellular health, developmental compe-
tence, and selective advantages has been acknowledged for
decades.
Platforms for metabolomic analysis are many and can be
categorized as nuclear magnetic resonance techniques, MS
techniques, and vibrational spectroscopy. Mass spectrometry
can be used alone or with other chromatographic analyzes,
including liquid chromatography, ultra performance liquid
chromatography, gas chromatography (GC), GC-time of
flight (TOF), capillary electrophoresis, and Fourier transform-
ion cyclotron resonance. Extensive review of methods,
strengths and limitations, and applications are available
in the following reviews [158,159]. Independent of the
methodology used in metabolomics analysis, there are
critical components to the process that can directly influence
the fidelity of outcomes and variability of results. First,
metabolism must be halted in biological samples and/or
biofluids at comparable times and as rapidly as possible
to terminate metabolic and enzymatic activities. This can
be accomplished with snap-freezing. Metabolites can be
isolated by numerous procedures that each have advantages
and disadvantages [160]. Platforms used for metabolic
assessments need to be unbiased, sensitive, selective, and
compatible with high-throughput analysis, while resulting
large data sets require powerful bioinformatics and statistics
that are amendable to providing adequate data analysis and
appropriate interpretations [156].
There have been numerous targeted and non-targeted
metabolomics approaches to address domestic animal
nutrient and seasonal impact on gamete/embryo metabolism,
metabolism during development, and metabolic requirements
for development [161–165]. While these studies have been
hypothesis-driven or focused on elucidating mechanistic
pathways in development, they have also given rise to tar-
geted—and to a greater extent, non-targeted—metabolomics
studies that attempt to predict oocyte/embryo quality. To date,
the majority of metabolomics studies related to predicting
and improving outcomes in human ART have been focused
on the preimplantation embryo. Although not the focus of
this paper, some of the outcomes of these human embryo
studies are informative about opportunities, as well as
limitations.
Numerous pilot, cohort, and case-controlled studies
have been performed regarding human preimplantation
embryo culture media metabolomics in predicting embryo
development, morphology, implantation, and successful
pregnancy [166–173]. As one might anticipate these reports
provided positive correlations, no correlations, and ultimately
collective non-conclusive guidance for the utility of embryo
culture media metabolomics in predicting ART outcomes.
There have been three randomized controlled trials (RCTs)
[174–176], which collectively concluded that metabolomics
(as performed in the trials) did not provide an objective
marker of embryo viability, did not demonstrate a benefit
in predicting a viable pregnancy, and did not significantly
improve selection of embryos in relation to live-birth rates
compared to embryo morphology selection alone. For more
detail on human embryo metabolic studies, one should
evaluate the review by Bracewell-Milnes and coworkers
[177]. The goals of this systematic review were to interrogate
published reports regarding metabolomics and human female
reproduction, with a specif ic focus on potential applications of
metabolomics of FF, embryo culture media, and endometrial
fluids in ART success.
To our knowledge, there is only a single peer-reviewed and
published report involving metabolomics profiling of human
oocytes [8]. The objectives of this study were to assess whether
near-infrared spectroscopy-generated metabolomic data col-
lected from short-term oocyte culture would correlate with
oocyte nuclear maturation and subsequent preimplantation
embryo development. In this case-controlled study, a total
of 412 oocytes from 43 patient ART cycles were denuded
of cumulus cell and placed into individual microdrops for
3–4 h prior to ICSI. Oocytes had different maturity (some
MI and some MII) and were followed in relation to day 3
and day 5 embryo development. The study authors concluded
that metabolomic profiling of spent media following short-
term culture of human oocytes can be performed, and is
(1) related to nuclear maturity, (2) able to predict embryo
development at both day 3 and day 5 stages, and (3) relates to
embryo viability. It is important to note that this study, like
many of the abovementioned human embryo studies, used
non-targeted metabolomics to gain metabolite fingerprints
with a computational categorization of profiles into “viability
indices” that may not necessarily be grounded in foundational
biology, development, and evidence-based predictions of cell
metabolism.
There are numerous extrinsic factors that can make
non-invasive assessment of the oocyte problematic. These
include non-oocyte cellular contamination, short culture
times, uncertainty of consistency of culture media and
media additives, the inf luence of interactive parameters
such as gaseous phase of culture, and human error inherent
to manual manipulations. While this study is interesting
and encouraging, additional studies are needed—especially
prospective RCTs. The above discussion also underscores
the need for well-controlled studies—not just in non-
targeted metabolomics, but also in targeted metabolomics.
Identification of predictive biomarkers of normal and
abnormal metabolism as it relates to proper and aberrant
human oocyte development and support of expected embryo
development is also wanting. Technical advances are being
explored in high-coverage targeted metabolomics [178]that
will likely advance the discovery of metabolic biomarkers.
The integration of automated, high-throughput targeted
metabolic assessments in real time, with microfluidics
platforms that are compatible with oocyte/embryo culture,
will be important in removing human technical variability
and standardizing assays and results. Multiple groups have
already demonstrated the ability to perform single embryo,
real-time metabolic assays assisted by microfluidic automated
fluid handling (Figure 1)[179,180]. It is quite easy to
recognize that multiple challenges exist in the application
of non-invasive oocyte metabolomics to predict ART
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8Non-invasive oocyte quality assessment
Figure 1. A Braille display-based microfluidic embryo culture and assay system that allows live-cell assessment of glucose consumption and lactate
production in real time. (A) Photograph of the whole system placed on a microscope. (B) Schematic cross-section of the cartridge on microscope. (C)
Schematic design of the embryo culture and metabolism assay cartridge. ITO (indium-tin-oxide heater is attached to the Braille displayed module to
maintain the media temperature at approximately 37◦C. (D) Graphic representation of real time on-cartridge measures of controls (media with 100, 50,
and 0 μM glucose) demonstrating the ability to quantify fluid glucose in real time on-cartridge with measure obtained ∼every 5 s and changing
concentrate every 24 min. (E) Not only was glucose consumption measured, but lactate production could be measured (picomoles/h). Lactate
production was measured from individual 2-cell mouse embryos (N=22) and individual mouse blastocyst (N=25) and levels measured in this
non-invasive manner, in real time on-cartridge, was similar to levels measured in the past with fluorescent microscopy (considered to be invasive if
performed on-cartridge). Figure adapted from [180] with permission from the Lab Chip, The Royal Academy of Chemistry.
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R. Sciorio et al. 9
outcomes, and even easier to accept the fact that advances in
many fields (biological and non-biological) and collaborative
integrations of techniques and expertise are needed to make
this vision come to fruition.
Oocyte novel imaging
Light microscopy and time-lapse imaging
The use of light microscopy is paramount to gamete and
preimplantation embryo assessment in the research and clini-
cal laboratory. The use of dissecting microscopes to find COCs
at retrieval is invaluable. Additionally, inverted phase-contrast
microscopy with Hoffman modulation or Nomarski differ-
ential interference allows for higher magnification and depth
of observation when assessing oocyte maturity, pronuclei for-
mation, and preimplantation embryo development. Recently,
time-lapse imaging of oocytes, zygotes, and embryos has been
investigated as a potential benefit in embryo selection by
morphometrics. Meseguer and coworkers demonstrated that
use of a time-lapse image acquisition system allowed determi-
nation of developmental events like the timing of cleavage and
suggested that using a multivariable model one could classify
human embryos with greater implantation potential [181].
Wong and colleagues reported that using non-invasive time-
lapse imaging of human embryos, one could predict blastocyst
development with developmental algorithms [182]. Chavez
and colleagues reported that similar time-lapse imaging sys-
tems could provide details in length of cell cycle progres-
sion and individual blastomere fragmentation that would be
predictive of embryo euploidy/aneuploidy [183]. While these
initial reports were exciting, subsequent larger, prospective,
and controlled studies have not necessarily confirmed such
findings. More recent reviews have concluded that evidence
does not support the routine use of time-lapse technology in
clinical ART laboratories and that future studies are needed
[184,185].
While the future use of time-lapse microscopy to assess
embryo developmental events and produce algorithms that
have predictive power remains a realistic research goal,
the use of time-lapse imaging or morphometrics as a non-
invasive microscopic imaging system for oocytes is wanting.
Beyond the phase contrast, optical imaging of oocytes without
cumulus cells to assess maturity (germinal vesicle intact,
metaphase I, or metaphase II), the utility of time-lapse
imaging of oocyte prior to insemination/fertility assessment is
questionable. Therefore, the evaluation of other microscopic
tools for oocyte assessment is required to determine if imaging
of oocytes can be developed as a non-invasive method for the
selection of oocytes with greater developmental competence.
Briefly, it is important to mention that light exposure to
oocytes and embryos can be detrimental to subsequent
development [186] and must be considered in all microscopy
evaluations (review see: [187]).
Polarized light microscopy
The use of polarized optics to evaluate gametes and embryos
is not a new idea and has been extensively reviewed else-
where [20]. Polarized light is a contrast-enhancing method
that yields quality images obtained by birefringent materials
that have structured organization. A goal of this, and other
imaging technologies discussed below, is non-invasive imaging
of intracellular organelle, structures, and/or processes that
provided informative guidance of selection or developmental
competence. In oocytes, polarized optics can allow visual-
ization of the spindle [188] and organization of the oocyte-
surrounding ZP [189]. One of the advantages of polarized
light microscopy is that it can be performed in real time and
on living cells. Clinical use of polarized optics with human
oocytes demonstrated that the metaphase II MS is not always
directly adjacent or beneath the extruded PBI [190]. This is
of potential interest, especially in oocytes used for ICSI, when
the alignment of the injection plane is based on the position
of the oocyte polar body. This observation also stimulates the
query of why and how does the extrusion of the first meiotic
polar body not align with the subsequent meiotic metaphase
spindle? This may likely be due to manual manipulation of
the oocyte with pipets during the removal of cumulus cells
in denuding manipulations—resulting in displacement of the
polar body in the PVS.
There have been studies focused on the use of polarized
optics to detect the presence or absence of the oocyte MS and
subsequent implantation and pregnancy rates. Unfortunately,
these studies have contradictory results [24,25]—indicating
that this live-cell imaging of the oocyte MS still warrants well-
controlled evaluation. These types of studies, as well as the
sensitivity of the oocyte MS to temperature as visualized with
polarized microscopy [191], emphasizes that the MS and the
tubulin polymerization is a dynamic structure/process. They
also stress the importance of considering time and space in
the dynamic evaluation of meiosis and the spindle. De Santis
and coworkers attempted to use spindle imaging via polarized
light microscopy as a predictor of oocyte quality [108], but
concluded that while there was a trend observed between
low oocyte spindle retardance and poor subsequent embryo
quality, the association was not statistically significant. What
may have been the most important report in this manuscript
was the observation that many oocytes that have extruded
the PBIhad not actually completed chromatin segregation
and were actually still in telophase I when one was able to
visualize the spindle. This observation could have significant
importance in considering the timing of sperm injection in
ICSI. If our light microscopy observations of the presence
of a polar body are not always indicative of completion of
meiosis I, progression to MII, and chromatic segregation,
then the injection process may be causing unrecognizable
damage to the telophase I spindle in the process of segregating
chromatin. This could be a future utility of polarized light
microscopy in non-invasive assessment of human oocytes with
a beneficial impact on subsequent normal fertilization and
euploid embryo development.
Raman microspectroscopy
This is a combination of Raman spectroscopy and confocal
microscopy and is founded on principles of inelastic scatter-
ing, which yield interactions of light and matter. The photon
scattering results in unique spectra that can be used to identify
molecules and their molecular bonds in living cells. We are
specifically discussing this technology over other technologies
such as multiphoton excitation fluorescence microscopy [192]
and light-sheet fluorescence microscopy [193], because of the
lack of labeled probes necessary for excitation, observation,
quantification. With that said, it is important to recognize that
these types of imaging systems (multiphoton and light-sheet)
are continuously developing and comparatively have distin-
guished themselves for having very low phototoxicity and
high spatial and temporal resolution—making them ideal for
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10 Non-invasive oocyte quality assessment
Figure 2. Composite schematic, micrographs, and graphic quantification of oocyte lipids with coherent anti-Stokes Raman scattering (CARS)
microscopy. (A) Schematic of CARS design. A single laser source provides a narrowband pump pulse (green) as well as a broadband Stokes pulse (red)
using a photonic crystal fiber (PCF). With a broadband source, two methods of detection were used for live-cell imaging: univariate photomultiplier tube
(PMT; requires less time to capture images) and multivariate charged-coupled device (CCD) CARS imaging. FI: Faraday Isolator, GP: Glan-Taylor Prism,
BP/LP/SP: band/long/short pass filter, DM: dichroic mirror, PCF: photonic crystal fiber. (B) Lipid content in mouse oocyte during meiosis as assessed and
quantified with CARS-PMT demonstrated that oocytes have low lipid content when they are GV-I meiotically incompetent (MeI). Oocytes gain lipids as
they grow and gain meiotic competence (still GV-I; MeC). As oocytes resume meiosis and progress from GV-I to metaphases II (MII), they lose
cytoplasmic lipids. (C) Oocytes from wild type and genetically obese (ob/ob) were assessed for lipid content with CARS-PMT. In meiotically competent
GV-I oocytes, oocyte lipid content was correlated with body mass. (D) Comparison of non-invasive lipid quantification with live-cell CARS-PMT to
invasive lipid quantification with oocyte fixation and staining with Nile Red. Micrographs represent both CARS-PMT (2850 cm−1) and NILE Red
fluorescence with their respective thresholded images for oocytes from human, mouse, cow, and pig. Graphically represented is quantification, showing
good agreement between CARS and fluorescence and expected higher intra-oocyte lipid in pig and cow compared to mouse and human. Scale bar is
50 μm. Figure adapted from [199] with permission from the Analyst, The Royal Society of Chemistry .
studies of development and cellular dynamics [194]. Raman
microspectroscopy has been used to evaluate mouse oocytes
[195]. These researchers found that using both synchrotron
Fourier transform-infrared and Raman microspectroscopy,
they were able to identify patterns of intracellular lipids and
areas of high protein content (assumed to be mitochondria)
that were different as oocytes matured. Such reports hold
promise as proof of concepts and require further investigation
to determine if Raman microspectroscopy will yield informa-
tive data in selecting oocytes beyond maturity, which can in
and of itself be assessed by standard light microcopy. Xenopus
oocytes have been evaluated using Raman microspectroscopy
to characterize the presence, absence, and cytoplasmic dis-
tribution of carotenoids [196]. An important component of
this investigation was the reported lack of detrimental impact
of Raman microscopy on Xenopus oocyte viability. Finally,
in a quite relevant report, Bogliolo and colleagues investi-
gated mouse aging and associated oxidative damage of MII
oocytes with Raman imaging. Using principal component
analysis, they showed distinguishable spectra between oocytes
from young mice versus those obtained from in vitro-aged,
oxidative-damaged, and old oocytes—specifically, significant
differences in lipid and protein components [197]. If this
held true for human oocytes, it could be an important non-
invasive means of oocyte assessment and selection. With
that said, although Raman spectroscopy is considered non-
invasive, the long imaging times required with spontaneous
Raman tend to counterindicate its use for live-cell imaging of
human gametes and embryos, thus potentially limiting its use
in clinical ART. Finally, Raman microspectroscopy has also
been used to evaluate oocyte responses to technical exposures,
such as vitrification and warming [198]. The sheep oocytes’
ZP carbohydrate secondary structures were altered by the
event of vitrification and warming, but not by cryoprotectant
exposure alone. These are interesting applications of Raman
imaging to address practical knowledge gaps at a molecu-
lar level. One would not be surprised to see future studies
using Raman imaging to gain non-invasive information about
oocyte selection.
A slight permutation of Raman microspectroscopy is
the use of coherent anti-Stokes Raman scattering or CARS
microscopy. Coherent anti-Stokes Raman scattering micros-
copy is considered a nonlinear vibrational microscopy
that has recently been demonstrated to allow non-invasive
quantification of oocyte lipids [199]. In relation to the
non-invasive nature of CARS, studies were performed to
assess CARS laser exposure (2–3 min) of mouse zygotes and
subsequent embryo development. As mentioned previously,
these types of experiments are essential to determine if the
technology is truly non-invasive and of potential clinical
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R. Sciorio et al. 11
relevance. It was found that the CARS laser exposure that is
compatible with intra-oocyte lipid quantification did not have
any detrimental effect on subsequent blastocyst development
rates (n=52 zygotes; 85%) compared to sham CARS laser
exposure controls (n=48 zygotes; 83%). Granted, this is
only a first step in determining the non-invasive influence of
CARS microscopy on embryo development in the mouse; it
also needs to be determined with human oocytes. Experiments
went on to demonstrate that the use of a broadband excitation
source allowed for single-wavelength images collected by
way of a photomultiplier tube (CARS-PMT) and could also
support the acquisition of the entire vibrational spectrum
for each pixel by way of charge-coupled device (CARS-
CCD). The CARS-PMT was shown to allow quantification
of intra-oocyte lipids similar to CARS-CCD (and Nile Red
staining as a control; invasive terminal experimental arm
where oocytes require fixation and staining); yet the CARS-
PMT required less time and laser exposure. Experiments then
demonstrated that intra-oocyte lipid quantification could be
performed on porcine, bovine, murine, and human oocytes
with CARS-PMT yielding expected species differences in
lipid droplet quantities (Figure 2A). Of interest was that
human oocytes assessed contained very few lipid droplets
in comparison to other species. What this might mean in
relation to human oocytes, energy stores in the form of
lipid droplets, and subsequent embryonic developmental
competence was discussed [199]. It was also demonstrated
that CARS-PMT could quantify statistically different levels of
lipid droplets in mouse oocytes of different meiotic stages
(growing meiotically incompetent germinal vesicle-intact
versus fully grown meiotically competent germinal vesicle-
intact versus metaphase II oocytes; Figure 2B). Finally, it was
also demonstrated that mice with different body composi-
tions (obese versus lean) had identifiable and quantifiable
differences in intra-oocyte lipid droplets (Figure 2C). While
these were first in field experiments that require repeating
and additional investigative queries to determine clinical
application, they are examples of the power of integrating
novel microscopic systems to gamete/embryo biological and
potentially practical studies. The authors concluded that
CARS-PMT appears to be a non-invasive live-cell imaging
technique of lipid quantification that (1) has experimental
power in basic cell biology, (2) may have practical utility
for identifying developmental predictive biomarkers while
advancing biology-based oocyte/embryo selection, and (3)
may have the ability to yield rational supporting technology
for decision-making in rodents, domestic species, and
human-assisted reproduction and/or fertility preservation.
Future experiments will examine the application of CARS
microscopy, as well as other novel means of viewing the intra-
structure, organization, and function of oocytes, to glean
biomarker data on oocyte developmental competences and
selection/deselection of oocytes in ARTs.
Conclusions and future directions in
non-invasive oocyte quality assessments
The integration of chemistry, genomics, microfluidics,
microscopy, physics, and other biomedical engineering
technologies into the basic studies of oocyte biology, and in
testing and perfecting practical solution of oocyte evaluation,
are the future for non-invasive assessment of oocytes.
Recent investigative and practical platforms for single oocyte
evaluations are in development (Figure 2)[200]. These will
likely be applied in the evaluation of oocyte transcriptomics
[201], single-cell polar body genomics [202], mitochondrial
genomics [203], metabolomics [180], and potentially non-
invasive genetic analysis of oocyte spent culture media similar
to current evaluations of non-invasive preimplantation genetic
testing for aneuploidy of embryos [204,205]. These goals are
not easy and have yet to be completely brought to fruition,
but progress is being made. It will be essential to foster and
maintain cross-discipline investigation in order to continue
building foundational, rigorous science that will determine
what represents a reliable biomarker of oocyte quality and
developmental competence, while in tandem developing
technologies and platforms that are truly non-invasive and
compatible with clinical laboratory utility in the realm of
ARTs. Basic and applied science are equally important in this
journey toward identification and application of non-invasive
oocyte assessment.
Acknowledgments
We would like to thank Mrs. Sarah Block for editorial comments. We
appreciate the content review and critiques by Dra. Martha Isolina
Garcia Amador. We acknowledge the American Society of Reproductive
Medicine Research Institute funding for partial support of manuscript
production. Finally, we would also like to acknowledge the scientific
contributions of many researchers in oocyte biology and assessment that
unfortunately have not been referenced in this review due to content and
size constraints.
Author contributions
RS, DM, and GDS conceptualized and wrote this manuscript.
Conflict of Interest
RS and DM have no conflicts of interest. GDS functions as a consultant
to Overture Life, a company focused on microfluidics and automation
of gamete and embryo isolation, manipulation, culture, assessment, and
cryopreservation.
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