Abstract efforts has been devoted to theAbstract efforts has been devoted to the



(Intra-cytoplasmic sperm injection) is a broadly utilized technique for
artificial fertilization. This approach has been successfully performed in
human oocytes as well as others such as mouse and bovine. The piercing through
the zona layer and the membrane needs to be achieved with a minimal biological
damage to facilitate a rapid healing. Since the injection methodology serves as
a crucial factor to success rate of ICSI, a significant amount of research efforts
has been devoted to the development of injections. In this review paper, we
summarize the major milestones for injection techniques in ICSI as well as a
comparative study of these techniques. Technical details are provided for each
milestone and each technique is evaluated from engineering perspective. At
last, we present a mechanism for healing process of membrane after drilling,
which could potentially provide guidance for improvement of injection method.

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ICSI, Injection, medical device, Piezo actuator, Ros-Drill, Piezo-ICSI.



As a broadly utilized technique for
artificial fertilization, ICSI (Intra-cytoplasmic sperm injection) has been
successfully performed in human oocytes as well as others such as mouse and bovine
1-6. Among the applications of ICSI, fertilization in mouse is of particular interest to
biological and biomedical research. The significance of biological cell
injection technology has attracted large amounts of research in an effort to
automate laborious cell injection tasks. For the previous decade, many research
efforts are dedicated to cell injection automation from a diverse array of aspects:
cell holding devices, cell injection method, vision-based automation, cell
injection process control, etc 7-8. Conventional ICSI employs a spiked
micropipette for facilitating penetration of the zona pellucida and piercing of
the oolemma to enable penetration into the ooplasm and injection of whole sperm
into the ovum. However, the elasticity of the mouse oolemma makes it very
difficult to penetrate without rupture, which causes irreversible damage to the
ovum 9-10,63. The piercing through the zona layer
and the membrane needs to be achieved with a minimal biological damage to
facilitate a rapid healing 9. A significant amount of research effort has
been devoted to the development of ICSI from perspective of injection


In conventional cellular injection
for ICSI, an individual oocyte is immobilized by a holding pipette with a
slight suction. The injection pipette containing the sperm head to be injected
into the oocyte is forced into the cell. The survival rates and fertilization rates of oocytes for
conventional ICSI vary from 80 to 90%, and 45 to 70%, respectively 11-16. Size
and sharpness of the needle used for injection are reported to be important for
success rate and among the crucial factors is injection method of the needle
into ooplasm 14.


Due to the low success rate and high labor intensity,
piezo-ICSI was firstly employed to mice in 1995 25-30,64-66, which achieved
high survival and fertilization rates in mice compared with those obtained by
conventional ICSI. Despite the
noticeably higher efficiency than conventional ICSI, in current practice,
piezo-assisted ICSI requires the presence of a small amount of mercury to
stabilize and suppress undesired lateral vibrations of the pipette tip under
mechanical impact 17-20. As of today, no other fluid material can replace mercury
which can provide sufficiently high density necessary to mitigate these
effects, preventing injection-induced damage to the ovum. Unfortunately, toxicity
of mercury prevents its massive deployment in labs and institutions. Over the
years, the alternative injection method had been desperately sought after to
eliminate dependence on toxic substance in ICSI.


Later, a novel, mercury-free, rotationally
oscillating drill (Ros-Drill) device was proposed as an alternative to
piezo-assisted ICSI. Since it doesn’t employ piezoelectric force actuator to
drive a micropipette for performing ICSI in the mouse, it does not require
mercury to suppress unwanted lateral motion of piezo. Preliminary results from
Ros-Drill in mouse embryos was reported to be comparable to those of
piezo-assisted ICSI using mercury. Moreover, minimal demand on human expertise
was claimed to be another benefit of this technology. These features result
from the computer automated nature of the Ros-Drill technology as explained in
the recent technology paper 21-23. However, there is very few experimental or
clinic data to support the statements and successful duplication by this method
is necessary.


Most recent improvements in ICSI
revolve around piezo-ICSI. Two main
directions for improvement are micropipettes design to improve fertilization
rate and injector design to avoid usage of mercury. For example, a piezoelectric
driven non-toxic injector for automated cell manipulation was proposed 24,
which claims to suppress detrimental lateral tip oscillation by centralizing
the piezo oscillation power on the injector pipette However, so far only
pipette movement is captured under high speed camera to show attenuation of lateral
movement. Experimental and clinic data is not available now. Capability of
piezo-ICSI may be compromised due to limiter applied in lateral dimension.


In this review paper, at first, we
summarize the major milestones for injection techniques in ICSI. Later their
comparisons are presented from extensive aspects. In essence, understanding of mechanism
of membrane healing is crucial in ICSI from two-folds: a. new injection method to
facilitate healing of membrane, b. facilitate healing after injection. At last,
we present a mechanism for membrane healing process after injection.



Major milestones

this section, technical details are provided for each milestone and each
technique is evaluated from engineering perspective.


2.1 Conventional ICSI

The first method for ICSI is performed manually and is known as conventional ICSI 25-35. With this
technique, the tip of the injection pipette is approaching gently towards the cell membrane about halfway or even much further into the oocyte. Then it is pushed against membrane rapidly
until it penetrates through the membrane. The spike-shaped pipette is most
commonly used for conventional ICSI 26. The high compliance of the mouse
membrane makes the penetration very difficult without rupturing the ovum 14. High
failure rate for conventional ICSI can be attributed to the damage caused by
the pressure of the injecting pipette on the cell membrane during the piercing
process. Damage needs to heal properly and rapidly; otherwise abnormal growth
occurs in the future stages of development 14,32,44,67. Since manual
operation can’t perform piercing process quickly and accurately enough, deformation
induced pressure inside membrane inevitably accumulates during piercing
process. In light of limitation in manual operation, injection parameters such
as injection depth (deformation level) and contact point become more critical
and stringent, which requires extensive study in membrane modeling and
intensive training in biologists and practitioners.

2.2 Piezo-assisted ICSI

The most popular procedure at the
present is the piezo-assisted ICSI which has proved to increase the success
rate beyond the conventional ICSI results 36-45. In this technique, to
generate minimal damage during the injection process, a piezo-activated axial
force is applied to the injecting pipette to pierce smoothly the zona pellucida
and then the cell membrane. Different from conventional ICSI, for
piezo-assisted ICSI penetration of the zona pellucida and oolemma can be
facilitated with a flat-tipped (not spiked) micropipette 38. In piezo-actuated
micromanipulation, the piezo-electric effect is employed to transmit a small
crystal lattice distortion to the tip of a pipette, moving it forward against
membrane in a prescribed manner. Piezo-actuated micromanipulation has multiple
applications in the study and engineering of gametes and embryos. For example,
it enabled the first intracytoplasmic sperm injection (ICSI) to produce mice,
the first nuclear transfer cloning of mice and pigs 41-42.


The typical
controller parameters of piezo-assisted ICSI are set to 10 V amplitude, 60s
duration, and 2 Hz frequency for mouse oocytes 36,46-48. Introduction
of piezo-actuation into ICSI for precise and consistent control is, in fact, a significant
progress towards an effective automated deployment of micro-injection operation. However, a small amount of mercury is usually
placed near the tip of piezo-actuated pipette to suppress its transverse
oscillations 17,18,42. This high intensity substance can effective suppress
vibration as the damping effect in lateral dimension.


The major supplier
for Piezo-actuated micromanipulation are Eppendorf PiezoXpert and Burleigh© Piezodrill.
Usually, their devices enable tuning of parameters,
such as speed and number of pulse and ramp-up/down slope for pulse 46,47.
These parameters need to be optimized after collections of experiments are
conducted to achieve good results.



The main operational difference
between piezo-drill and Ros-Drill is in the motion generation methods.  Instead of the axial impact type action in
the piezo-drill, the Ros-Drill rotationally oscillates the micropipette at a
selected frequency and amplitude 21-22. The intention for Ros-Drill design is
that the ideal straight micropipette would rotationally oscillate about its
axis without a lateral motion. However, the eccentricity in pipette due to
manufacturing tolerance would generate some whirling motion during oscillation 21-22.
Therefore, the eccentricity level determines the unwanted lateral movement.


In alpha version of Ros-Drill, to
avoid excessive lateral displacements, pipette is oscillated with very small
angular amplitude (1°
peak-to-peak) and at frequencies that above natural frequencies of mode 1 and
mode 2 (90-100 Hz) and much lower than the 3rd mode 21. 30° bent
pipettes are selected for during injection. This Ros-Drill microinjector
prototype employs a PLC (Programmable Logic Controller) as the digital
controller, which has a maximum sampling frequency of 1 kHz 22. This
constraint limits the controllable trajectory to be at the maximum frequency of
500 Hz due to the Nyquist sampling rule. A DC servo motor
is employed to actuate the pipette to track a sinusoidal position reference.
The servo motor comes with an optical encoder with 512 lines and quadrature
signature capability which could only provide a sensor resolution of 0.176° 22.


Most recently reported ICSI tests
use injection pipette oscillations up to 0.2° amplitude and maximum frequency of
500 Hz 22. Small angular oscillation is to minimize excessive lateral motion.
High frequency is adopted to avoid cell membrane to follow the stimulus,
facilitating a clean piercing process. One assumption is made in this new
technique that the motion generated from piezo-actuator is faithfully
transmitted to tip of pipette due to high rotational stiffness of the pipette
holder and the pipette. Experiments were done to show that Ros-Drill operation generates
smaller lateral oscillations at the tip, compared with the piezo-assisted ICSI
cases 21-22. Although biological results based on this prototype are very
promising, the trajectory tracking performance of the alpha prototype is not
satisfactory due to low-resolution of position sensor and low control sampling
rate of the PLC.


Beta version Ros-Drill was designed
and prototyped to be a high-precision, compact, and inexpensive setup. Main
improvements in hardware lie in microcontroller with sampling frequency of
10kHz and rotational motion sensor with resolution of 0.09° 49-51.
Despite the improvement in hardware, resolution of encoder is still very low
compared with target amplitude tracking (0.2° amplitude). This is a typical
mechatronic design with resource constraint, which stems from the position
sensor in terms of size, resolution and costs demanded by the application 52-54.
magnitude control is at the core of precision control and presents no
particular difficulty when it uses appropriate sensors 55. In order to track
a 0.2° amplitude oscillation with sensor
with 0.09° resolution, conventional control
techniques fail to achieve desired tracking capability. Initially, a look-up
table based gain scheduling technique was proposed to transform this
low-resolution application to conventional proportional-integral-derivative
(PID) method 49-50. To counteract model uncertainty and disturbance in the
control process, an adaptive control technique was designed to guarantee
desired performance by adjusting control gains 56-57. Moreover, an effective
and systematic methodology which can achieve zero magnitude error tracking
control for extremely low-resolution encoder was designed and implemented 58-61.
Essentially it treats a stochastic stroke control problem by converting it into
a deterministic one. The proposed control strategy consists of a two-stage
adaptive control logic which adapts the control gains to achieve the actual
peak-to-peak stroke. Comparisons of alpha and beta version of Ros-Drill are
shown in Table 1.


Table 1: Comparisons of alpha and
beta version of Ros-Drill.


Sampling rate (kHz)

Encoder resolution (deg)

Control algorithm

Trajectory tracking capability
(amplitude in deg)

Alpha Ros-Drill






Beta Ros-Drill




Adaptive control



technique looks promising from drilling mechanism and implementation
perspective. Somehow, very few institutions or clinics follow this protocol and
very limited experimental data is available.


2.4 Enhanced piezo-assisted ICSI

2.4.1 Micropipettes improvement

Material and geometry of
micropipettes also plays an important role in piezo-assisted ICSI. Researches
and studies are conducted for optimization of geometry of micropipettes. It is shown
that the Piezo-ICSI using micropipettes with a wall thickness of 0.625 ?m significantly
improves the survival, fertilization, good-quality day-3 embryo, pregnancy, and
live birth rates when compared to the conventional ICSI and Piezo-ICSI using
micropipettes with a wall thickness of 0.925 ?m 62. 


2.4.2 Piezoelectric driven non-toxic

In piezo-drill cell injection, mercury is placed at the
tip of pipette to reduce generation of lateral tip oscillations of injection
pipette. Some alternative is proposed to suppress this excessive lateral motion
in piezo-driven cell injection. A new piezo-driven cell injector design
centralizes the piezo oscillation power on the injector pipette which
eliminates the vibration effect on other parts of the micromanipulator 24.
Detrimental lateral tip oscillations of the injector pipette are attenuated to
a desirable level even without the help of mercury column. This mercury-free
injector can sublime the piezoelectric driven injection technique to completely
non-toxic level 24.


Essentially, the new injector employs a rigid
attachment to limit lateral motion from piezo actuator. The efficacy of this
limiter is demonstrated by the suppression of lateral movement under high speed
camera by 50% 24. However, no experimental or clinical data is available for
this newly proposed method. From engineering perspective, additional constraint
on actuator in one dimension usually compromises actuation capabilities of
other dimensions due to mechanical coupling. Piercing force from axial
oscillation of piezo-electric device is inevitably attenuated as a consequence.
More simulation/modeling, e.g. finite element analysis and experiments need to
be performed to demonstrate efficacy of this technique.


Methodology comparison

section provides comparison of three major injection methods, i.e.
conventional, piezo-assisted and Ros-Drill, as shown in Table 2. Conventional
injection is manual and labor-intensive compared with piezo-assisted and Ros-Drill.
From actuation perspective, piezo provides axial vibration whereas Ros-Drill
rotational oscillation. In spite of availability of commercialized medical
device for conventional ICSI, satisfactory success rate still calls for
intensive training in biologists and practitioners. Advanced control algorithm
and mechatronic design enable Ros-Drill to achieve desired motion at low cost,
which puts Ros-Drill at a favorable position in contrast to Piezo-assisted
device. The statistics for publications pertinent to injection methods of ICSI
are based on data from 1995 to 2017.


Nowadays, based on number of related
publications and deployment, piezo-assisted method dominates in experimental
and clinic activities in ICSI. In terms of survival and fertilization rate, Ros-Drill
and Piezo-assisted method are comparable. However, since much more experimental
data is available for Piezo-assisted method than Ros-Drill, the survival and
fertilization rate for
Piezo-assisted method is more statistically significant. Again Ros-Drill
technique look promising from drilling mechanism and proposed implementation
perspective. Somehow, very few institution or clinics follow this protocol and
very limited experimental data is available.

2: Comparisons between three major injection techniques for ICSI.



Survival rate (%)

Fertilization rate (%)

Cost ($k)


# Publication (1995-2017)

# Usages




45 to 70


labor intensive












Rotational oscillation




insufficient data




Mechanism of membrane recovery after drilling

In essence, understanding of
mechanism of membrane healing is crucial in ICSI from two-folds: a. new
drilling method to facilitate membrane
healing, b. method to facilitate healing after drilling.


The cell membrane, also called plasma membrane,
is very thin with thickness of a few molecules.
The major role of cell membrane is to isolate the intracellular contents. In
addition, the cell membrane also serves to maintain the shape of the cells.
Another function of the membrane is to control intercellular transport via
endocytosis and exocytosis. The cargo lipids and
proteins are moved from the cell membrane as substances are internalized
through endocytosis whereas vesicles containing lipids and proteins fuse with
the cell membrane to cargo in important proteins and enzymes. The cell
membrane primarily consists of a mix of membrane proteins and lipids. The occupation of lipids can range from 20 to 80
percent of the membrane, relying on the cell type and their roles. Lipids help
to maintain membranes flexibility, membrane proteins regulate the cell’s
chemical environment and promote the transfer of molecules via the membrane.
Phospholipids are a major component of cell membranes. Phospholipids form a bilayer where their hydrophilic head
areas are oriented to the extracellular fluid, whereas their hydrophobic tail stay
away from the cytosol and extracellular fluid. The lipid bilayer is semi-permeable.
It allows limited molecules to diffuse through
the membrane. Cholesterol is another major lipid component of cell membranes.
Cholesterol molecules are largely distributed between membrane phospholipids to
prevent cell membranes from stiffening through keeping phospholipids from
being packed together. Glycolipids present
on cell membrane surfaces with function of helping the cell to identify other
cells of the body. The cell membrane
contains two distinct types of associated proteins as well. Peripheral membrane proteins are
exterior to the membrane. They are connected to membrane by binding with other
proteins. Integral membrane
proteins are inserted into the membrane, exposing on both sides of
the membrane. Cell membrane proteins have various functions. Structural proteins play an
essential role in the cell support and shape. Cell
membrane receptor proteins is
vital for cells communication with their external environment through the use
of hormones, neurotransmitters, and
other signaling molecules. Transport
proteins of globular proteins, transport molecules across cell membranes
through diffusion.  Glycoproteins are embedded in the
cell membrane with a carbohydrate chain attached to them, which contributes the
cell communications and molecule transport across the membrane.

The membrane does not contribute to
the cell’s mechanical strength, which is attributed to the cytoskeleton or cell
wall. In addition to
the lipid bilayer and integral proteins, RBC membrane possesses a 2D
cytoskeleton tethered to the lipid bilayer. The RBC membrane cytoskeleton has a
2D six-fold structure, and is made of the spectrin tetramers connected at the
actin junctional complexes, forming a 2D six-fold structure. The cytoskeleton
is connected to the lipid bilayer via the “immobile” band-3 proteins at the
spectrin-ankyrin binding sites and the glycophorin protein at the actin
junctional complexes.


can heal after drilling. Once the micropipette is withdrawn, the lipid molecules rearrange themselves to reduce exposure of
their hydrophobic regions to water. However, the rearrangement of the lipids at
the pore boundary results in configurations that are not as energetically favorable as the
bilayer itself, such that there is an energy penalty for the formation of
holes.  This energy penalty can be described by a line
tension or edge tension, which is an energy per unit length along the
boundary of the hole. The effective edge tension is temperature-dependent and
vanishes at sufficiently high temperature, where
the membrane is unstable against hole formation
even in the absence of mechanical stress. Therefore, it drives the lipid molecules to the
hole to reduce the line tension until the membrane is healed. The healing
process is highly dependent on the mechanical state of the membrane. When the
membrane is under swelling (hydrated), it becomes thinner and its hydrophobic
core is increasingly exposed to water so it ruptures, releasing tension through
increasing the hole such that the cell becomes ruptured.  On the other hand, when the membrane is under
compression (dehydrated cell), the lipid molecules tend to migrate to the hole
to release the compression and thus accelerate the healing the membrane.
Therefore, we hypothesis that the dehydrated examined cells will facilitate healing after drilling.
Our above discussions are based on the analysis of the mechanics of the lipid
bilayer and cytoskeleton. We hope that these discussions can potentially
stimulate and steer new experiments in this area.



5. Conclusion

A significant amount of research
effort has been devoted to injection method for ICSI to achieve minimal
biological damage to facilitate a rapid healing of membrane. Major milestones
for injection techniques in ICSI is summarized. Their comparisons are also
presented from engineering perspective. Piezo-assisted method is dominant in
ICSI activities, around which further improvement in ICSI is built. Ros-Drill
technique looks promising from drilling mechanism and proposed implementation
perspective. However, very few institution or clinics follow this protocol and
very limited experimental data is available. At last, we present a mechanism
for healing process of membrane after drilling, which could potentially provide
guidance for improvement of injection method.