851765

research-article2019
JLAXXX10.1177/2472630319851765SLAS TechnologyWeng

Review
SLAS Technology

IVF-on-a-Chip: Recent Advances in

2019, Vol. 24(4) 373­–385
© 2019 Society for Laboratory
Automation and Screening
Microfluidics Technology for In Vitro DOI: 10.1177/2472630319851765
https://doi.org/10.1177/2472630319851765
journals.sagepub.com/home/jla

Fertilization

Lindong Weng1

Abstract
In vitro fertilization (IVF) has been one of the most exciting modern medical technologies. It has transformed the landscape
of human infertility treatment. However, current IVF procedures still provide limited accessibility and affordability to most
infertile couples because of the multiple cumbersome processes and heavy dependence on technically skilled personnel.
Microfluidics technology offers unique opportunities to automate IVF procedures, reduce stress imposed upon gametes
and embryos, and minimize the operator-to-operator variability. This article describes the rapidly evolving state of the
application of microfluidics technology in the field of IVF, summarizes the diverse angles of how microfluidics has been
complementing or transforming current IVF protocols, and discusses the challenges that motivate continued innovation in
this field.

Keywords
PDMS, lab on a chip, intracytoplasmic sperm injection, embryo culture, cryopreservation

Introduction According to the Centers for Disease Control and

In vitro fertilization (IVF) is a complex series of procedures
used to treat infertility or genetic problems and assist with
the conception of a child.1 It is the most effective form of
assisted reproductive technology (ART). During IVF,
mature oocytes are retrieved from the patient’s ovaries and
fertilized by a sperm in a lab. Then the fertilized oocyte is
implanted into the patient’s uterus. Current IVF procedures
include the selection of motile sperm, retrieval and process-
ing of oocytes, insemination, in vitro embryo culture, and/
or cryopreservation. Two main approaches of insemination
in the lab are conventional IVF2,3 and intracytoplasmic
sperm injection (ICSI).4 During conventional IVF, each
single oocyte is placed in an oil-covered microdrop in a cul-
ture dish in the presence of an optimal concentration of
sperm, and fertilization occurs when one sperm penetrates
the oocyte naturally. ICSI, on the other hand, is a technique
in which a single sperm is injected directly into the oocyte’s
cytoplasm through a thin needle for fertilization. ICSI has
been the dominant treatment for almost all forms of male
infertility, and it has also been increasingly adopted to over-
come fertilization failure.4 In 2012, for example, ICSI was
used in 93.3% of fresh IVF procedures (i.e., using non–
freeze-thawed embryos) to treat male factor infertility and
was used in 76.2% of all fresh IVF procedures with and
without male factor infertility.5
Prevention, a total of 182,111 IVF procedures with the
intent to transfer at least one embryo were performed in the
United States in 2015, contributing to 1.7% of all infants
born in the United States during the same year.6 Despite its
immense potential of overcoming human fertilization fail-
ure, IVF treatment still remains inaccessible and unafford-
able to the majority of infertile couples around the world,
especially in resource-limited countries and areas. Current
IVF procedures suffer from a variety of limitations,7 such as
being costly and time-consuming.8 The IVF procedures also
rely heavily on highly skilled embryologists, which often
results in variation from operator to operator and a lack of
standardization.7
However, we can gain leverage on addressing the above
challenges facing IVF applications by incorporating minia-
turization and automation technologies that have emerged
in recent decades, such as microfluidics. Microfluidics
1
enEvolv, Inc., Medford, MA, USA
Received Feb 20, 2019, and in revised form April 20, 2019. Accepted for
publication April 30, 2019.
Corresponding Author:
Lindong Weng, enEvolv, Inc., 200 Boston Ave #2975, Medford, MA
02155, USA.
Email: l.weng@enevolv.com
374
technology manipulates and controls fluids of microliters to
picoliters within a network of channels with dimensions
from tens to hundreds of micrometers. Microfluidics has
many unique advantages, including reduced reagent vol-
ume, shorter reaction time, and the scalability for parallel
operation.9 Miniaturization and automation offered by
microfluidics can further improve the precision of experi-
ments, lower the limits of detection, and allow performance
of techniques and experiments that are usually not possible
on the macroscale.10
Over the past two decades, microfluidics technology has
demonstrated great potential for streamlining IVF proce-
dures.11 Microfluidics provides an alternative to almost
every single step in the process of IVF, including but not
limited to improved selection of motile sperm, automated
processing of oocytes, in vitro follicle growth, embryo cul-
ture, and cryopreservation. Furthermore, the capability of
reproducing a given single IVF procedure on an isolated
microfluidic module offers the opportunity to investigate
and understand the reproductive physiology in each step of
assisted human reproduction.
In the literature, there have been several outstanding
reviews discussing the applications of microfluidics in
assisted human reproduction.7,8,11–13 However, the strong
synergy between microfluidics and IVF has been constantly
driving the evolution of concepts, designs, methodology,
and applications in the field. Many new microfluidic
approaches were proposed and investigated in the past 2 y,
which has brought unprecedented outcomes or served the
unmet needs for automating IVF procedures.14–25 This article
reviews recent progress in employing microfluidics to pro-
cess gametes, perform IVF, and/or culture embryos. It also
discusses the state of the art of microfluidics-facilitated
cryopreservation of gametes and embryos for IVF. In the
end, several challenges and opportunities are outlined to
motivate the continued innovation of microfluidics with
implications for IVF.
Improved Selection of Motile Sperm
on a Chip
The fundamental challenge of sperm selection for IVF is
dictated by biology: a heterogeneous population of ∼108
sperm per milliliter with a short lifetime in vitro. Traditional
sperm selection methods such as swim-up and density gra-
dient centrifugation are not only time and labor intensive
but also result in low yield of motile sperm or high DNA
fragmentation.26 To address these challenges, microfluidics
has been integrated with procedures such as semen analysis
for male infertility diagnosis27,28 and sperm selection based
on motility14,29–33 and/or facilitated by chemotaxis,15,34,35
rheotaxis,36–38 thermotaxis,15,39 or inertial focusing.40
Asghar et al.31 developed a sperm-sorting device in which
only the most motile and functional sperm could pass
SLAS Technology 24(4)
through a polycarbonate membrane filter against gravity and
swim into a retrieval reservoir on top of the filter, leaving
dead and less motile sperm behind (Fig. 1A,B). This device
enabled the sorting of an unprocessed semen sample with
higher DNA integrity and fewer reactive oxygen species
than traditional sperm-sorting methods. Nosrati et al.32 pre-
sented another microfluidic device to select sperm based on
the progressive motility in 500 radial microchannels (Fig.
1C). It took only one step to purify and select the sperm of
high DNA integrity from 1 mL of raw semen within less than
20 min. Compared with traditional practices of either swim-
up or centrifugation, experiments with bull sperm indicated
more than 89% improvement in selected sperm motility and
clinical tests with human sperm showed more than 80%
improvement in DNA integrity. In addition to the motility-
driven mechanism, Li et al.39 used the principle of thermo-
taxis to evaluate human sperm on a microfluidic device
facilitated by an interfacial valve. The temperature gradient
was established and accurately controlled by an external sys-
tem. Thermal-responsive human sperm were found to have a
tendency to accumulate in the higher-temperature area in the
four tested temperature ranges. The developed device
trapped the thermotactic sperm fraction in a discrete branch
by an air-liquid interfacial valve.
Chinnasamy et al.14 developed a microfluidic device,
named the Simple Periodic ARray for Trapping And isola-
tioN (SPARTAN), in which they designed an array of pillars
to efficiently isolate motile and morphologically normal
sperm from raw semen with lower epigenetic global meth-
ylation (Fig. 1D–F). This device modulated the directional
persistence of sperm, increasing the spatial separation
between progressive and nonprogressive motile sperm pop-
ulations within 10 min compared with 60 to 90 min with the
traditional swim-up technique. The proposed microfluidic
method yielded significantly reduced DNA fragmentation
(SPARTAN: 4%–6% vs. swim-up: ∼13%), improved mor-
phology (SPARTAN: ∼52% vs. swim-up: ˜24%), and nuclear
maturity (SPARTAN: ∼44% vs. swim-up: ∼26%).14
Furthermore, Nagata et al.25 developed a diffuser-type
microfluidic sperm sorter to select functional and progres-
sively motile bovine sperm with high DNA integrity (Fig.
1G). The microchannel geometry was optimized to gener-
ate the fluid dynamics that are relevant in the propulsion of
sperm. This study identified the fertile subpopulation to be
the sinuous, transitional cohort based on the kinetic and tra-
jectory patterns. The findings contrasted with the conven-
tional belief that the fertilizing sperm are always vigorously
motile and more linear. This microfluidic approach also
demonstrated the possibility of using visual pattern recogni-
tion as a realistic means of predicting fertilizing ability
without subjecting sperm to tedious assays.
Using human and bovine semen, Zaferani et al.24 demon-
strated a different high-throughput microfluidic device that
could passively isolate motile sperm within corrals inside a
Weng 375

Figure 1. Recent microfluidic systems for sorting motile sperm. (A, B) A membrane filter-embedded microfluidic device for
sorting of motile sperm. (A) The microfluidic device contains an inlet, a filter, and two poly(methyl methacrylate) (PMMA) chambers.
(B) Illustration of the working principle: raw semen sample is injected from the inlet. Two PMMA chambers are separated by a
membrane filter. The most motile sperm swim up through the filter, leaving unhealthy and dead sperm in the bottom chamber. The
figure is adapted from ref. 31 with permission from John Wiley & Sons, Inc. (C) A microfluidic sperm selection device with capacity
for 1 mL of raw semen. (i) Schematic view of the device, featuring a bottom layer with a network of 500 radial microchannels and
a top layer with 1.45-mm depth at the injection ring and 0.8-mm depth at the outlet chamber. (ii) Schematic view of the assembled
device and close-up view of the microchannel vertical walls. (iii) One milliliter of raw semen is injected around the ring, and live
and motile sperm navigate through high-viscosity fluid toward the central outlet, resulting in the collection of ∼100,000 sperm for
assisted reproductive technology. The figure is adapted from ref. 32 with permission from the Royal Society of Chemistry. (D–F)
A three-dimensional periodic array of pillars for sperm selection. (D) A photo of the microfluidic device (scale bar: 10 mm). (E) A
scanning electron microscopy image of the 30 × 26 µm periodic pillar array (scale bar: 50 µm) and a zoom-in image (inset) showing
micropillars in detail. (F) Illustration of the microfluidic pillar array and sperm traveling through the array. The figure is adapted from
ref. 14 under the Creative Commons Attribution license. (G) A diffuser-type microfluidic sperm sorter device. (i) Microchannel design
and geometry from the bottom view of the device. The medium flowing out from chamber A is supplied into chamber B and C. Motile
sperm are sorted from frozen-thawed semen in chamber B and accumulated and collected in chamber C. (ii) Close-up illustration
of the microchannel network showing the junction area as indicated by the red square. Inset: A photo of the device composed of
polydimethylsiloxane (PDMS) and glass. The figure is adapted from ref. 25 under the Creative Commons Attribution license. (H)
A microfluidic corral system separating sperm based on rheotaxis. (i) Side view of the sperm in the vicinity of the top surface. The
sperm tail experiences a greater force than its head. (ii) Top view of the sperm. The rotation caused by shear leads to an upstream
orientation. (iii) PDMS microfluidic device featuring seven corrals inside a flow channel. The figure is adapted from ref. 24, complying
with the license for Proceedings of the National Academy of Sciences of the United States of America articles.
376
fluid channel, separating them from the rest of the diluted
sample (Fig. 1H). Computational simulation revealed that
certain flow rates could create a rheotaxis zone in front of
the corral for sperm to move upstream or downstream
depending on their motility. For example, motile sperm that
passed through these regions would reorient themselves and
begin swimming toward the opposite direction of the flow
until they entered the corral and became trapped. Thus, pro-
gressively motile sperm were separated from the rest of the
sample. Taking advantage of this rheotactic behavior of
sperm, which was tunable with the injection flow rate, this
microfluidic device was able to corral motile sperm with
progressive velocities in the range of 48 to 93 µm/s and 51 to
82 µm/s for bovine and human samples, respectively. More
importantly, the separated fractions of both human and
bovine samples featured 100% normal progressive motility.
It is challenging to isolate the most motile and presumably
healthiest sperm from semen samples that have low sperm
counts and/or low sperm motility. Microfluidic systems dis-
cussed previously have shown potential to sort motile sperm
with flow systems. However, the small field of view (FOV)
of conventional microscopes commonly used to image sperm
motion limits the number of sperm cells that can be tracked
simultaneously.29 To address this challenge, Zhang et al.29
integrated a lensless charge-coupled device with a microflu-
idic device to enable wide FOV and automatic recording as
the sperm moved inside a microfluidic channel. The inte-
grated system enabled the sorting and tracking of a popula-
tion of sperm within a microfluidic channel. This channel
was able to be monitored in both a horizontal and vertical
configuration similar to the swim-up column method. Sperm
motility was quantified by tracing the shadow paths for indi-
vidual sperm. Moreover, motile sperm that reached the outlet
were conveniently retrieved from the channel at the end of
the process. This technological approach was suggested to be
highly useful for isolating motile sperm from a pool of a
small number of sperm confounded by low sperm motility.29
Microfluidics is also a powerful tool to understand how
sperm compete for insemination of the oocyte in vivo. Very
recently, Zaferani et al.17 revealed that sperm with the high-
est motility were most likely to make it through the narrow
junctions inside the female reproductive tract, by using an
“hourglass” shape microfluidic device to simulate the fluid
mechanical properties of these narrow junctions in vitro.
These pinch points were found to prevent sperm with a
motility below a certain threshold from advancing through
the pinch point to the fertilization site, as the slower sperm
accumulated in front of the pinch point and were caught in
oncoming currents.
Microfluidics-Facilitated Processing
of Oocytes
In the ovary, individual oocytes are natively surrounded by
cumulus cells (specialized granulosa cells), forming an
SLAS Technology 24(4)
organized structure known as the cumulus-oocyte complex
(COC). The cumulus cells that are in close contact with the
oocyte, also known as corona cells, develop cytoplasmic
projections that cross the zona pellucida and form gap junc-
tions with the oolemma.41 In vivo, the cumulus cell mass is
naturally removed from the oocyte upon fertilization.
Following conventional IVF, the embryo has to be denuded
from the cumulus cell mass within the first 24 h because the
by-products from the interaction between hyaluronidase
and cumulus cells can damage the zona pellucida and
decrease embryo viability.42 Furthermore, oocytes must be
denuded before ICSI to allow the evaluation of oocyte mor-
phology including the nuclear maturation stage41 and facili-
tate the injection of a sperm.43 In IVF clinics, oocytes are
usually denuded from the cumulus cell mass via a combina-
tion of enzymatic action of hyaluronidase and mechanical
pipetting, which are inefficient and suffer from operator-to-
operator variation. However, microfluidics technology has
helped automate the denudation procedures, which has the
potential to reduce time and cost as well as improve
standardization.
Zeringue et al.44,45 developed a microfluidic device to
mechanically remove the cumulus cell mass from single
bovine zygotes (Fig. 2A,B). The zygote is the first stage of
the early embryo, following the fusion of the haploid male
and female pronucleus. In the proposed device, the cumulus
cell mass was partially removed and reoriented after the
cumulus-zygote complex passed through a constricted
channel that was only slightly narrower than the complex.
Then, two suction ports, which were significantly narrower
than the complex, were used to remove the remaining
cumulus cell mass. This device processed one zygote at a
time and required manual controlling and switching of mul-
tiple fluid flows to achieve positioning and movement of
the cumulus-zygote complex.
Very recently, Weng et al.16 developed a microfluidic
device to denude mouse oocytes from the surrounding
cumulus cell mass, facilitating the evaluation of oocyte
quality and the injection of sperm for ICSI (Fig. 2C,D).
Hyaluronidase-treated COCs, at either the germinal-vesicle
or metaphase II (MII) stage, passed through a series of
jagged-surface constriction microchannels of optimized
­
geometries (Fig. 2E). The jagged inner wall of the constric-
tion channels facilitated stripping off the cumulus cell mass.
Denudation efficiency of more than 90% was achieved
when processing COCs through at least 100 repeats of
expansion (500 µm long and 500 µm wide) and constriction
(640 µm long and 90 µm wide) units at a flow rate of 1 mL/
min. Oocytes that were denuded by the device showed com-
parable fertilization and developmental competence com-
pared with the traditional mechanical pipetting technique.
In the manual denudation process, about 10 COCs were
first incubated with 0.3 mg/mL hyaluronidase for 5 min and
then underwent a series of manual pipetting through a glass
capillary that had an inner diameter of 125 µm. In addition,
Weng 377

Figure 2. Current microfluidic systems for processing of female gametes and zygotes. (A, B) A microfluidic device to remove
cumulus cell mass from a zygote. (A) The main channel features conditioning regions and removal ports. Inset: The funnel-shaped
inlet well. (B) Four main stages in the cumulus removal process. (i) Cumulus cell mass is reoriented as it passes through the second
constricted region. (ii) The complex is at removal port 1. (iii) The complex rotates as it moves from port 1 to port 2. (iv) Remaining
cumulus cell mass is removed at port 2, yielding a clean zygote. The figure is adapted from ref. 44 with permission from Springer
Nature. (C–E) An on-chip oocyte denudation microfluidic device. (C) A schematic diagram of the repeating constriction-expansion
channels. (D) The operation of the oocyte denudation device involves a syringe pump applying a constant flow and a Tygon tubing
preloaded with cumulus-oocyte complexes suspended in the medium. Inset: A photo of a polydimethylsiloxane device bonded to a
glass microscopic slide. (E) Moments of oocytes traveling through the jagged-surface constriction microchannels as they are denuded
gradually. The figure is adapted from ref. 16 with permission from the Royal Society of Chemistry. (F, G) A constricted microfluidic
channel to study oocyte deformation. (F) A photo of the microfluidic device. (G) Time sequence images of oocytes deformed
in a constricted channel of 50 µm wide and 150 µm high under a flow rate of 10 µL/min. The figure is adapted from ref. 46 with
permission from Springer Nature.
378
computational simulations in this study revealed that on-
chip denudation was able to generate a smaller shear than
the manual denudation method and thus impose less
mechanical stress on the cells. The flow rate in manual
denudation was estimated to vary between 500 and 750 µL/
min.16 As a result, shear stress may fluctuate between pipet-
ting cycles by up to 250 Pa in manual denudation. Therefore,
another advantage of the proposed device was to maintain a
constant shear stress profile due to the constant flow rate
applied.
While the oocytes travel through microchannels, mechan-
ical stress arises from the physical contact between the cells
and channel walls, which may damage the fertilization
capacity of processed oocytes. Luo et al.46 studied the defor-
mation of mouse oocytes under shear flow and its subse-
quent impact on their spindle structure as the oocytes
traveled through constricted microfluidic channels of dif-
ferent sizes at various flow rates (Fig. 2F). They found that
oocytes were significantly deformed and their spindles
were severely damaged (characterized by immunocyto-
chemistry staining of the microtubules and chromosomes)
under relatively high flow rates (e.g., 10 µL/min through a
constricted channel of 50 µm wide and 150 µm high), as
seen in Figure 2G. Although shear may help remove cumu-
lus cells from the oocytes for denudation, the shear stress
generated in microfluidic channels must be controlled care-
fully by optimizing the geometry and flow rate to preserve
the structure and functionality of oocytes.
Taking advantage of the correlation between oocyte
quality and its sedimentation rate, Iwasaki et al.22 developed
a microfluidic device to separate high-quality bovine
oocytes to improve the conception rate of IVF. Their device
featured a long separation channel of 10 mm long, 700 µm
wide, and 1 mm high. After denuded oocytes were injected
into the separation channel, they traveled toward the outlet
at an optimal flow rate controlled by a syringe pump and
settled gradually. The outlets of the separation channel were
divided into upper and lower reservoirs. High-quality
oocytes settled faster than poor-quality ones in a phosphate-
buffered saline buffer with 0.05 wt% polyvinyl alcohol and
0.3 M sucrose. Thus, high-quality oocytes were collected
from the lower outlet reservoir. IVF results showed that the
developmental rate of blastocysts of oocytes collected from
the lower outlet (36.0%) was significantly higher than those
collected from the upper outlet (14.1%).
In Vitro Growth of Ovarian Follicles on
a Chip
In vitro culturing of ovarian follicles provides a promising
strategy to restore fertility for female patients facing prema-
ture infertility due to cancer therapies. Woodruff and
coworkers47–49 developed a three-dimensional (3D) culture
system to support in vitro development of follicles,
SLAS Technology 24(4)
producing mature oocytes of fertilization capacity. Immature
follicles were encapsulated within sterile alginate beads,
which maintained the oocyte’s 3D architecture and cell-cell
interactions.47–49 On the other hand, microfluidics approaches
of encapsulation of ovarian follicles for 3D culture have
also been reviewed previously.50 A flow-focusing microflu-
idics technique was used to generate core-shell droplets
encapsulating Peromyscus early preantral follicles.50 The
collagen core and alginate shell of these hydrogel droplets
mimicked the cortex and medulla, respectively, reproducing
the native milieu in a mammalian ovary, as mechanical het-
erogeneity was found to be critical in regulating follicle
growth.50 The results showed that 5 MII oocytes out of 11
COCs (45.5%) were obtained by culturing early preantral
follicles in the optimal biomimetic ovarian microtissue with
a 0.5% collagen core and 2% alginate hydrogel shell.
Moreover, these MII oocytes and in vitro maturation of the
COCs could be further activated to obtain two-cell stage
embryos.
Xiao et al.51 developed a microfluidic system to support
mouse ovarian follicles to produce the human 28-d men-
strual cycle hormone profile, which controlled human
female reproductive tract and peripheral tissue dynamics in
single, dual, or multiple unit microfluidic platforms. Mouse
ovarian tissue was cultured on a microfluidic platform
based on pneumatic actuation, simulating the in vivo female
reproductive tract and the endocrine loops between organ
modules including ovary, fallopian tube, uterus, cervix, and
liver.51 This microfluidic platform achieved organ-organ
integration of hormonal signaling and pregnancy-like endo-
crine loops and also presented a powerful tool for drug dis-
covery and toxicology research.
IVF and Embryo Culture on a Chip
An integrated microfluidic system has the prospect of
achieving IVF and embryo incubation with reduced human
intervention, high efficiency, and minimal operator-to-oper-
ator variation. Suh et al.52 demonstrated in vitro insemina-
tion, co-incubation, and fertilization of murine oocytes in a
microfluidic chamber. This microfluidic system featured a
barrier gate that allowed only media and sperm to flow
through, whereas the oocyte was blocked without deforma-
tion. Such selective passage resulted in the increase in the
local sperm concentration around the trapped oocyte.
Therefore, the sperm-oocyte interaction was increased
compared with the microdrop-in-a-dish technique. This
study demonstrated that a smaller total number (i.e., 1000–
4000) and a lower concentration of sperm (i.e., 2 × 104 to 8
× 104 sperm/mL) were required to achieve a similar fertil-
ization rate to the corresponding control using the micro-
drop-in-a-dish technique, which required an optimal
concentration of 1 × 106 sperm/mL. Similarly, using a geo-
metrical constraint to block the oocyte within a microfluidic
Weng
chamber, Clark et al.53 demonstrated that the incidence of
polyspermy IVF of porcine oocytes was statistically signifi-
cantly lower (p < 0.05) than the oocytes fertilized in the
traditional microdrop system, whereas the sperm penetra-
tion and male pronucleus formation rates were comparable.
The flow velocity and profile of sperm traveling through
this microfluidic chamber were investigated in a follow-up
study by Lopez-Garcia et al.54 It was suggested that the
sperm-oocyte interaction could be controlled by regulating
the local flow rate and adjusting the channel geometry,
thereby further reducing polyspermy by maintaining a low
sperm concentration around the trapped oocyte.
Han et al.55 developed a microfluidic chamber featuring
an array of microwells that could facilitate single oocyte
trapping, fertilization, and embryo culture (Fig. 3A). With a
comparable fertilization rate to the conventional microdrop-
in-a-dish technique, this microwell array has the potential
of simplifying oocyte handling and manipulation, allowing
rapid and convenient medium changing, and enabling auto-
mated tracking of any single-embryo development. In
another study, Angione et al.56 engineered a microfluidic
device with hydrodynamic traps to longitudinally monitor
viable, separate single oocytes, including perfusion of stim-
ulation media, exchange for immunolabeling reagents,
introduction of sperm for insemination, and prolonged
incubation at controlled temperatures.
Furthermore, Ma et al.57 developed a different microflu-
idic device that integrated each step of IVF, including
oocyte positioning, sperm screening, fertilization, medium
replacement, and embryo culture. Individual oocytes were
positioned in a 4 × 4 array of octa-column units (Fig. 3B).
The array was connected to four straight channels, forming
a cross with the oocyte positioning array in the center,
allowing efficient motile sperm selection and facilitating
rapid medium replacement. The fertilization process and
early embryonic development of the individual zygote were
recorded and analyzed by in situ fluorescent staining. By
passing through the screening channels, the average mouse
sperm motility increased from 60.8% to 96.1%. The embryo
growth rate and blastocyst formation were similar between
the microfluidics group and the conventional microdrop
group. The healthy blastocysts developed in the microflu-
idic device could be conveniently retrieved and transferred
using a pipette. Heo et al.60 developed another embryo cul-
ture and assay device to perform automated periodic analy-
sis of embryo metabolism, such as measuring the
time-dependent nutrient consumption by single or multiple
mouse blastocyst-stage embryos with pmol/h sensitivity.
This microfluidic system represented another compelling
case for integrated microfluidic platforms to perform practi-
cal single-embryo culture and real-time biochemical analy-
sis, which has the potential of improving the success in
clinical ART laboratories by screening high-quality
embryos.
379
Once the embryo is formed, it takes nearly a week to
travel down the fallopian tube to enter the uterus. While it
travels in the oviduct, the fertilized egg is subjected to
dynamic physical stimulation such as muscular contrac-
tion.61 Kim et al.61 developed a microfluidic in vitro culture
system for bovine embryos, in which peristaltic muscle
contraction was mimicked by using a partially constricted
channel. The system also generated a gravity-driven
dynamic flow using a micro-modulated tilting machine.
The results showed that the proportion of eight-cell devel-
opment among total embryos in the constricted channel
(56.7%) more than doubled that in the straight channel
(23.9%), indicating the vital role of constriction in the early
development of bovine embryos after IVF.
Role of Microfluidics in
Cryopreservation of Gametes
and Embryos
Cryopreservation of gametes and embryos is highly desir-
able for many reasons, primarily to store excess genetic
material and to control the timing and precision of embryo
transfer.62 As IVF was introduced in the clinics to treat
human infertility, cryopreservation of embryos using slow
freezing and rapid thawing with dimethyl sulfoxide
(Me2SO) was soon applied to human embryos in the early
cleavage stage and led to the first live birth from cryopre-
served human embryos.63 Cryopreservation of human
embryos has now become a routine procedure in ART clin-
ics.64 Also, when single men or women require urgent can-
cer treatment such as chemotherapy, they are at risk of
losing their fertility. In such cases, cryopreservation of gam-
etes may be the only option for future restoration of repro-
ductive potential.63
Slow freezing and vitrification are two main approaches of
cryopreservation. Slow freezing is a classic preservation
method for living cells in which the sample is frozen at an
optimal rate. The freezing rate is selected such that it is slow
enough to avoid detrimental intracellular ice formation but is
fast enough to limit solute toxicity due to excessive dehydra-
tion in the presence of extracellular ice.65 Alternatively, vitrifi-
cation is an ice-free process in which the liquid is cooled
rapidly to outrun ice crystallization and transits into a non-
crystalline solid state (a glass) below the glass transition tem-
perature,66 which typically requires a combination of an
ultra-rapid cooling rate and a very high concentration of glass-
forming solutes (which can themselves be toxic), although
vitrification with low concentrations of cryoprotective agent
(CPA) were also reported in the literature.67,68 During thaw-
ing, the rewarming rate must be sufficiently high to prevent
devitrification, especially in large-volume samples.69
To help the preserved gametes or embryos overcome the
detrimental stresses induced by cryopreservation, the
380 SLAS Technology 24(4)

Figure 3. Recent microfluidic systems for manipulation or cryopreservation of oocytes/embryos. (A) A microwell-structured microfluidic
device for oocyte trapping, in vitro fertilization, and embryo culture. (i) The top microchannel layer features a microchannel of 1 mm wide
to transport sperm and oocytes and a microchamber of 3 mm wide to accommodate all the oocytes in the microwells. (ii) The bottom
microwell layer features 200 mm × 200 mm square microwells. (iii) Cross section of the double-layer microfluidic device. (iv) Image
of the microfluidic device bonded to a glass microscopic slide. (v) View of the microfluidic device with the top layer lifted. The figure is
adapted from ref. 55 with permission from the Royal Society of Chemistry. (B) A microfluidic in vitro fertilization device. (i) The device
includes four sperm inlet pools and an oocyte/embryo positioning well. The pools and the well are connected by sperm-screening channels.
(ii) Scanning electron micrograph of the 4 × 4 array of the octa-column unit designed in the positioning well. (iii) Optical image of a
representative octa-column unit. (iv) Illustration of a representative octa-column structure. The inner radius is 200 µm, whereas the outer
radius is 300 µm. The gap (g) between the two adjacent columns is 50 µm. (v) Side view of the microfluidic device. The figure is adapted
from ref. 57 with permission from the American Chemical Society. (C) A microfluidic perfusion system to characterize the transport
properties of human oocytes. (i) An expanded view showing the configuration of the microdevice with a microfluidic perfusion chamber
and an integrated temperature-controlled stage. (ii) Diagram showing the connections between the microdevice and three syringes and the
microchannels. The arrows indicate the direction of fluid flow. The figure is adapted from ref. 58 with permission from the Royal Society of
Chemistry. (D) A digital microfluidic device for embryo vitrification. (i) Device structure and assembly. (ii) Regions for vitrification medium
dispensing and loading/extraction of embryo. The figure is adapted from ref. 59 under the Creative Commons Attribution license.
Weng
addition of CPA, such as Me2SO and glycerol, has become
routine. CPAs can depress ice nucleation, promote glass
transition, help preserve the integrity of plasma membranes,
or stabilize the native structure of proteins.70 Permeable
CPAs, including alcohols, sulfoxides, and amides, can read-
ily penetrate the cell membrane by diffusion, acting in both
extra- and intracellular spaces.
When exposed to a hypertonic solution of a permeable
CPA, cells initially lose and then uptake water in response
to the osmotic gradient, accompanied by a constant influx
of CPA. As a result, cell morphology undergoes a shrink-
and-swell behavior.71 Oppositely, a swell-and-shrink behav-
ior is observed when permeable CPAs are removed from
freeze-thawed cells before fertilization or transplantation.
Such abrupt volumetric changes can potentially collapse or
explode the cells that are subjected to cryopreservation,
especially for large cells such as oocytes and embryos.
Therefore, it is critical to understand the transport of water
and solute across the membranes of oocytes or embryos and
then optimize the loading and unloading processes of CPAs,
such as the number of addition and/or removal steps and the
increment of concentration in each step.
In recent years, a number of microfluidic perfusion
chambers have been developed specifically for the mea-
surement of cell membrane permeability.58,71–73 Heo et al.73
proposed a microfluidic device for the quantitative mea-
surement of oocyte volume during various CPA loading
protocols. Stepwise, linear, or complex CPA concentration
profiles were created to load CPAs into a trapped oocyte,
and the oocyte volumetric response to each profile was
measured. With both linear and complex profiles, 1.5 M
propanediol could be loaded into the oocyte in less than 15
min with a volumetric change of less than 10% compared
with the isotonic condition. Very recently, Zhao et al.58
designed a perfusion chamber with a “road block” to hold
individual oocytes in place and coupled the chamber with a
temperature-controlled stage to measure the osmotic behav-
ior of human oocytes at 4, 15, and 25 °C (Fig. 3C). Because
the proposed device was not made of polydimethylsiloxane
(PDMS), the fabrication did not require soft lithography
and plasma treatment. However, the assembly of multiple
components, including the perfusion chamber, temperature-
controlling element, sealing gasket, and thermocouple, may
still demand great care. Very recently, Chen et al.74 demon-
strated the correlation between the membrane permeability
of oocytes and their fertilization and developmental compe-
tence. When comparing the membrane permeabilities of
high-quality (blastocyst rate: 60%) and low-quality (blasto-
cyst rate: 0%) oocytes that were determined on a microflu-
idic device, the authors found that high-quality oocytes
were more responsive to hypertonicity, yielding less signifi-
cant volumetric shrinkage than low-quality ones.
Despite the characterization of membrane permeability
of gametes and embryos to various CPAs, the attempt to
381
eliminate osmotic stress is still limited by the practicality of
performing numerous precise pipetting steps in a short
period of time. Miniaturization and automation offered by
microfluidics provide a promising solution to overcome this
limitation. Lai et al.75 developed an automated CPA addi-
tion protocol based on microfluidics to minimize the
osmotic stress during the vitrification of bovine oocytes or
murine zygotes, yielding better morphology, higher cell
quality, and improved developmental competence than CPA
exposure using the manual pipetting method that is cur-
rently used in clinics. In the proposed device, a number of
individual oocytes or embryos were held within the expo-
sure chamber by suction, being exposed to a continuous
temporal gradient of CPA concentration that was generated
by synchronizing two programmable syringe pumps that
were connected to the microfluidic device.75 In another
study, a digital microfluidic device was developed to auto-
mate the preparation of embryos for vitrification (Fig.
3D).59 Digital microfluidics has been a useful tool for
sequential sample processing, enabling the manipulation of
liquid droplets over an array of insulated electrodes.76
Droplets on the digital microfluidic platform acted as
micro-vessels to move an embryo and introduced it to a
series of cryoprotectants of different concentrations, stimu-
lating a stepwise CPA addition protocol. The results demon-
strated that the embryo survival and developmental rate
achieved by using this digital microfluidic approach was at
least comparable with that of the manual operation based on
the limited sample size tested. This digital microfluidics
approach achieved automated operation, generation of
cryoprotectant concentration gradient, and loading and
retrieval of embryos. More recently, Zheng et al.77 devel-
oped a microfluidic system for CPA loading and unloading
that was further integrated with ultra-rapid cooling and
rewarming protocols for cryopreservation of human umbili-
cal vein endothelial cells. Their CPA loading/unloading
device involves only a flow-focusing section and a mixing/
equilibrium region and is easy to use and relatively high
throughput; it also generates less osmotic, toxic, and
mechanical stress than traditional pipetting techniques.
We have seen a growing number of microfluidics devices
developed in the field of cryopreservation during the past
two decades.78–81 Although many of these devices were
designed for other types of cells, some of them may be
adapted relatively easily to facilitate the cryopreservation of
gametes and embryos. Several representative examples are
discussed in detail below.
Deutsch et al.82 developed an Individual Cell-based
Cryo-Chip (i3C) made of mold-injected polycarbonate and
BK7 glass. The device featured an array of picowells, each
of which was designed to maintain an individual cell in
position during cryopreservation and accompanying treat-
ments. More than 97% of the cells tested (human promono-
cytic U937) were found to retain their position in the
382
picowells throughout the entire freeze-thaw cycle and
medium exchange. This device enabled the comparison
between prefreezing and postthawing data at single-cell
resolution. In a subsequent study, Afrimzon et al.83 demon-
strated that the functional activity of U937 cells that were
freeze-thawed on the i3C devices had no statistically sig-
nificant difference from the control cells that were cryopre-
served via conventional cryo-vials. Most importantly, the
combination of the multiparametric analysis at a single-cell
resolution and the optical and biological features of the
device enabled the accurate determination of the functional
status of individual cells and subsequent retrieval and utili-
zation of the most valuable cells, which are highly desirable
for on-chip cryopreservation of oocytes or embryos.
As mentioned above, vitrification is an ice-free cryo-
preservation approach in which the aqueous phase is cooled
rapidly to enter the glassy state in the absence of ice both
inside and outside the cells. A major challenge to cell vitri-
fication is intracellular ice formation induced by devitrifica-
tion (i.e., formation of visible ice in a vitrified sample)
during rewarming of the vitrified cells at a moderate rate
while they are still at cryogenic temperature.67 Therefore,
high concentrations (up to 8 M) of permeable CPAs and
small sample volume (∼2.5 µL) have to be used to facilitate
vitrification during cooling and minimize devitrification
during rewarming, yielding high toxicity and limiting scale-
up capacity. Huang et al.67 revealed that hydrogel microen-
capsulation could effectively inhibit devitrification during
rewarming. Cells were encapsulated into alginate micro-
droplets using a nonplanar microfluidic device. Their results
showed that if ice formation was minimized during cooling,
intracellular ice formation became negligible in hydrogel
microencapsulated cells during the entire cooling and
rewarming cycle. The proposed approach enabled vitrifica-
tion of mouse embryonic stem cells and human adipose-
derived stem cells with low concentration of permeable
CPAs (1.5 M 1,2-propanediol) and nonpermeable CPAs
(0.5 M trehalose) in a relatively large volume (up to 250 µL)
using conventional plastic straws.67 It is worth mentioning
that these plastic straws are also the commonly used closed
system for the vitrification of oocytes and embryos.84 Very
recently, alginate hydrogel microencapsulation and Fe3O4
nanoparticle-mediated rewarming were leveraged to sup-
press ice formation during the vitrification of mesenchymal
stem cells in polytetrafluoroethylene capillaries, presenting
another promising approach to achieve low-CPA (∼2.5 M)
and large-volume (10 mL) vitrification.85
Summary
Although it has significantly transformed the landscape of
treating human infertility, IVF remains inaccessible and
unaffordable to the majority of infertile couples, especially
those living in remote, resource-limited regions. This is
SLAS Technology 24(4)
mainly due to its complicated, error-prone procedures and
strong dependence on technically skilled personnel. During
the past two decades, microfluidics technology has demon-
strated its potential of automating IVF procedures, reducing
stress on gametes and embryos, and improving standardiza-
tion. Although the state of the application of microfluidics
in IVF has been rapidly evolving, future challenges and
opportunities still exist for microfluidics to advance our
understanding of assisted human reproduction and optimize
IVF protocols in clinical settings.
Most of the microfluidic systems were designed to specifi-
cally handle either male or female samples or to focus on the
postfertilization stages such as embryo culturing. However,
multimodular microfluidic systems hold the promise of fully
automating and standardizing clinical IVF procedures, which
would alleviate the dependence on human intervention and
reduce the financial burden on patients. Despite recent
advances,57 further development of such IVF-on-a-chip sys-
tems may draw synergistic support from technologies such as
organ-on-a-chip,86 3D printing,87–89 and paper-based micro-
fluidics.90,91 Also, microfluidic devices that mimic the in vivo
structure of reproductive tissues and organs will offer more
accuracy and flexibility when coupling different modules that
represent different IVF procedures.
Most of the microfluidic devices fabricated for IVF so far
are made of PDMS. However, this type of transparent and
gas-permeable elastomer potentially has negative effects for
cell studies at the microscale, such as deformation, evapora-
tion of culture medium, molecular absorption, leaching of
un-cross-linked oligomers, and hydrophobic recovery.92
Other biocompatible materials may be superior when used
for handling gametes and embryos. Thermoplastics such as
poly(methyl methacrylate), polystyrene, and cyclic olefin
copolymer have emerged as alternatives to manufactured
microfluidic devices for biological applications.93 For
example, cyclic olefin copolymers are a relatively new
group of polymers with desirable properties,94 such as
chemical resistance, excellent optical transparency, nonab-
sorption of small molecules, and biological compatibil-
ity.95,96 Moreover, fabrication techniques such as
micro-milling, hot embossing, and injection molding are
available for the production of microfluidic devices made
of cyclic olefin copolymers to overcome the bonding
issues.92 Berenguel-Alonso et al.97 have fabricated two
microfluidic devices using hot embossing and micro-
milling on cyclic olefin copolymers. One device was used
to trap and fertilize bovine oocytes, and the other was used
to store sperm and assess its quality over time.
There are relatively fewer microfluidic systems16,46,98
that can be used to facilitate the preparation and quality
evaluation of oocytes for IVF in comparison with those
used for sorting sperm or culturing embryos. In fact, the
current clinical procedures used for female patients demand
a great amount of intervention from well-trained
Weng
embryologists, limiting the accessibility and affordability
of IVF treatment. Therefore, an all-in-one microfluidic sys-
tem is highly desirable to enable automation of the proce-
dures, including oocyte retrieval from follicular fluid,
selection of high-quality oocytes, and/or oocyte denudation
for ICSI. To echo the first challenge discussed above, it
would also be interesting to integrate oocyte-processing
devices with those handling sperm or embryos to strengthen
the prospect of IVF-on-a-chip.
The application of microfluidics in cryopreservation has
been largely limited to the characterization of biophysical
properties of gametes and/or embryos. Other microfluidic
devices are suggested to meet the clinical interests more
closely, such as freezing or vitrifying gametes or embryos on
a chip. In particular, the miniature nature of microfluidics has
the advantage of handling extremely small volume of sam-
ples, which may provide favorable characteristics to facilitate
ice-free vitrification if materials of sufficiently high thermal
conductivity are used in device fabrication. Further integra-
tion of microfluidics with cryopreservation has the potential
to support automation and reduce user-specific variability,
enhance discovery of new CPA exchange protocols, and
improve outcomes in assisted human reproduction.99
Acknowledgments
The author would like to gratefully thank Dr. Edward Kai-Hua
Chow for the invitation to contribute this review article to the
journal.
Declaration of Conflicting Interests
The author declared no potential conflicts of interest with respect
to the research, authorship, and/or publication of this article.
Funding
The author received no financial support for the research, author-
ship, and/or publication of this article.
ORCID iD
Lindong Weng https://orcid.org/0000-0003-4811-6531
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