Microfluidic technology for in vitro fertilization (IVF)
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With the recent development in science and technology, the advancement in in vitro fertilization to treat infertility has been one of the most revolutionary advances. However, the success rate of the IVF process depends on the efficiency of each step of the process. The physical tools used to enhance the process continue to change. Microfluidics technology is an emerging technology being used in multiple biological applications for the miniaturization and specification of laboratory techniques. Technology is used along with IVF to enhance the outcome by facilitating every step of the process. Microfluidics can be used to handle gametes, culture embryos, cryopreservation, and for many other applications. This review will highlight the applications of microfluidics in different stages of IVF, including the handling of gametes, sperm collection and isolation, sperm sorting, embryo culture, cryopreservation and the fabrication process of microfluidics, focusing on the benefits and shortcomings of these applications.
KeywordsCryopreservation Embryos Gametes In vitro fertilization (IVF) Microfluidics
The advancement made in IVF is probably the most exciting scientific development for infertility treatment. It has offered hope for couples who cannot normally bear children since the first IVF child was born in the Royal Oldham Hospital, London, UK in 1978 . Since then, interest in IVF has grown. With increasing medical technologies, scientists have been able to study the process of IVF extensively, and the careful attention given to every step has definitely improved the fertilization rates.
While the intention of IVF was to solve the problem of infertility, a problem arose when couples with faulty sperm quality and quantity could not undergo the process of IVF. To solve this problem, a new process has been introduced in which a single spermatozoon is injected directly through the zona pellucida of the oocytes . The process is termed intracytoplasmic sperm injection (ICSI). The development of the ICSI process has made fertilization possible even in severe cases of compromised sperm properties . In the years between 2008 and 2010, of the 4.4 million assisted reproductive technology (ART) processes initiated, 1.1 million babies were born . The use of these types of ART continue to increase with increasing problems of infertility due to environmental, occupational, and other hazards.
Since 2010, various approaches have been taken to improve IVF. The microfluidic technology is integrated in each step of the IVF process, because the success rate of IVF depends on each step of the process. Microfluidic technology has so far been one of the best technologies developed for the advancement of this field. The technique miniaturizes and simplifies the long hectic procedure of ART in a simple chip. The ART field has certainly been enhanced with its introduction. However, a limitation of this technology remains.
Even though a great deal of research has been conducted in this area, microfluidics is seldom used on a daily basis in clinics worldwide, because of its complexity and unconvincing results related to the human model. This lack of application needs to be overcome, and laboratory technicians need to be open-minded about adopting the new technology to replace the conventional technology.
In this review, we will highlight the uses and benefits of microfluidics in the different steps of in vitro fertilization along with its drawbacks and possible solutions.
2 In vitro fertilization (IVF)
IVF and ICSI are the earliest developed artificial insemination techniques . The process of IVF has not been able to fulfill the demand of infertile couples when the male partner shows subfertility. To overcome this shortcoming, the ICSI process was introduced in 1987 . During this process, the best sperm is selected which is mechanically introduced to the oocyte. The process is also used when the female egg cannot be hatched easily. The first successful birth using this process was in 1992 . The success rate of ICSI depends on the motility of fresh retrieved or thawed sperm and on the maturity of the sperm selected . Experiments conducted by Park et al., and Shibahara et al., separately showed better fertilization and pregnancy rates using fresh motile sperm [12, 13]. Behavioral and psychological differences were not observed in the offspring conceived with IVF/ICSI technology . However, both IVF and ICSI children can show some chromosomal abnormalities compared to the naturally conceived children [15, 16].
The entire treatment process with ART is burdensome, sometimes resulting in patients discontinuing the treatment. To minimize this obstacle, all the procedures must be made as short and as efficient as possible. Today, the process has become somewhat less tedious, with the introduction of microfluidic technology.
Microfluidic technology, which was first introduced in the early 1990s, is defined as the study of the behavior, precise control, and manipulation of fluid in microenvironments. The first microfluidic device was miniaturized gas chromatography (GC) developed at Stanford University . Since then, this technology has been used in different fields such as chemistry , molecular biology, and developmental biology , although it is most widely used in the biomedical field for the control of fluid transport in the cell analysis system, drug delivery system, and for assisted reproductive technology [19, 20, 21, 22]. It is an emerging field used for the miniaturization and simplification of laboratory techniques based on chips with fabricated microchannels and chambers . Beside these fields, it is also used in forensic science . Its use in these areas has provided numerous benefits overall due to its decreased cost in the manufacture, use, analysis, disposal, etc.
2.1.1 Fabrication of microfluidic device
Previously, glass materials and silicon were the commonly chosen materials as the basic substrates for the development of microfluidic devices. Glass material has the best biocompatibility and has a high temperature resistance and solvent compatibility [25, 26]. However, it was fairly expensive to use in larger amounts . Also, a clean room was mandatory for its production, the sealing process was time consuming, and the yield was low . The use of polymer instead of glass and silicon as a substrate for fabrication was introduced in the late 1990s . The polymers used in microfluidic technology can be categorized into polydimethylsiloxane (PDMS) and thermoplastics. Both PDMS and thermoplastics have shown biocompatibility with many biomolecules and cells [30, 31]. Polymers such as poly(dimethyl siloxane) (PDMS), and poly(methyl methacrylate) are less expensive and easier to manipulate than silicon glass . Nowadays, polymers are commonly used since they are simple, inexpensive, and readily disposable . Among all the polymers, PDMS is the most commonly used and most preferred due to its elasticity, transparency, gas permeability, and nontoxic nature [28, 34]. In addition, in polymers such as PDMS, the channels can be formed by molding or embossing rather than etching, while the devices can be thermally sealed using adhesives due to which, PDMS has been shown to be safe when used as a substrate for reproductive cells . However, more care is needed to control the surface chemistry of PDMS to avoid sample absorption, evaporation, and leaching, while PDMS is mostly incompatible with organic solvents .
Pamela et al. demonstrated the applications of different types of polymers in microfluidic technology . PDMS is a frequently used material in microfluidics as it is inexpensive and has a simple fabrication process. However, PDMS is comparatively soft, rendering it difficult to resist higher pressure . An experiment conducted to find an alternative material to PDMS resulted in the development of thermoset polyester (TPE), which has surface stability as well as good chemical and solvent compatibility [42, 43].
Fabrication of micro-particles by droplet microfluidics has shown great potential in areas like cell biology, drug delivery and also in biosensors . Droplet-based microfluidics has grabbed attention due to its refined control over the flow of multiple flows in the microscale. Also, the fabrication of dielectrophoretic (DEP) microfluidic device used in IVF has followed a similar fabrication method as explained before. Thus, the IVF biochip was proved to enhance the rate of in vitro fertilization .
With the development of 3D printing technology, the fabrication process of the microfluidic polymer was utilized, rendering it a popular prototype for microfluidic device fabrication. With its easiness in fabrication of complex microfluidic devices and its cheap price, 3D printing has shown dramatic growth and interest within the microfluidic community for the past few years . However, no printer type is perfect which questions the reliability of the print size of microfluidic channels with correct dimension in a required sized device. Drawbacks of 3D printings are not only limited to this but also extend to surface properties and compatibility issues .
However, various studies are being performed to address the improvements needed at every step of the process, which guarantees a future for microfluidic polymer in this field.
2.2 IVF with microfluidics
As a relatively new technology in the field of ART, microfluidics is attracting a great deal of attention . Microfluidic insemination could increase the fertilization rate in cases of low sperm concentration (0.01–0.08 × 106 sperm/cell) compared to the conventional method . Microfluidics application in the IVF process is continuing to increase. Maturation, insemination, and oocyte manipulations along with other processes using microfluidic devices are now fairly common in IVF. For example, the technique of removing the cumulus cell at the zygote stage using the microfluidic microchannel has been developed [50, 51]. The same device was used for the removal of zona pellucida by washing a plug of lysing agent over the embryo .
A large amount of research has been undertaken in the field of IVF with microfluidics and is ongoing for enhancing the process.
Similar to IVF, ICSI has been the choice of many patients with male infertility . However, while the threat of oocyte lysis while inserting the needle into the oocyte still remains , the causes of oocyte degeneration have seldom been investigated . One of the reasons for oocyte degeneration is technician error . During the ICSI process, for fertilization, a single sperm is injected into the oocyte obtained after maturation . Gonadotropin is important for controlled ovarian stimulation, as it helps to produce the optimum number of oocytes . ICSI should only be used in cases of severe male infertility, because there is less evidence to confirm the effectiveness of ICSI on other non-male factors of infertility [58, 59]. The microfluidic device has been proven to be helpful in reducing the time required for sperm concentration in poor quality semen samples in ICSI treatment . Microfluidic devices have also been reported to be useful for ICSI treatment in human assisted technology to increase sperm concentration in poor quality semen samples .
While the human ICSI process is widely used and successful, microfluidics can provide greater accuracy than that provided by a human technician and requires less time than human labor. However, the method can be comparatively expensive. For future use, the process needs to become more economical and should maintain similar or improved results with more consistency.
2.2.1 Sperm collection and isolation with microfluidics
Many microfluidic techniques have been developed in the field of ART, such as the manipulation, counting, and sorting of sperm . Depending on the sperm characteristics (semen volume, sperm motility, sperm viability, sperm morphology, etc.), either sperm isolation or manipulation is selected. Also, depending on the sperm characteristics, the conventional IVF or ICSI is performed. Various steps are involved in sperm processing for IVF, including media washing, semen overlay and swim up of sperm out of the seminal plasma, and density gradient centrifugation, or a combination of the above methods . (http://apps.who.int/iris/handle/10665/44261). These processes are known to enhance the efficiency of the process. However, centrifugation is said to cause sub-lethal damage to the sperm . Oxidase stress produced when the sperm DNA is exposed to a high level of reactive oxygen species during centrifugation is correlated with sperm DNA damage [63, 64, 65]. It has been reported that the sperm DNA damage has a negative impact on artificial reproductive technology [66, 67]. Attempts have been made to develop devices that isolate the sperm without the need for centrifugation [68, 69, 70]. Conventional sperm preparation techniques such as swim up, density gradient centrifugation, and glass wool filtration are now obsolete, since they cannot produce the expected sperm populations for ART .
The microfluidic technique is said to obtain high motility, enhanced percentage of normal morphology, and significantly reduced percentage of DNA-damaged sperm in comparison with the centrifugation process during semen processing . A microfluidic device was recently developed that selects sperm based on their progressive motility in 500 parallel microchannels and uses a one-step procedure for semen purification and high-integrity DNA sperm selection . The device can select 1 ml of a sample in less than 20 min.
It is found that the maximum DNA fragmentation is found in the patient with recurrent pregnancy loss (RPL). During the early stage of the development of microfluidic devices for sperm counting, fluorescence was used to count the sperm . Various microfluidic applications were later added that used electrical impedance and other technologies such as oriented sperm swimming technology to count the sperm . From an experiment conducted by Ainsworth et al., the electrophoretic system was developed for the separation of spermatozoa, which resolved the problem of DNA damage .
2.2.2 Sperm sorting
Also, Huang et al. developed a laminar stream-based microfluidic device which could separate human motile sperms and flow cytometry analysis was used to enhance the sperm motility sorting efficiency . An experiment was performed by Shao et al. in which optical trapping was used to quantify the motility of the sperm and to select the sperm . Likewise, Tsai et al. discovered a microfluidic device for sperm sorting that can sort the motile sperm in 30 min and with better DNA integrity than prior to sorting . It was also observed that the microfluidic sperm sorting system could reduce the treatment time for intracytoplasmic sperm injection [3, 60].
2.2.3 Embryo culture with microfluidics
The embryo growing naturally and that growing in the laboratory differ, since inside the mammalian body, the embryo develops in a fluid environment within the oviduct (fallopian tube) and uterus. This is termed ‘dynamic environment’ and could be one of the reasons for the differences in fertilization. To address the gap in the knowledge of the environmental conditions of the oviduct and uterus during in vitro embryo culture, sequential culture media processes have been attempted . Conventionally, gametes and embryos are cultured in different culturing dishes with different laboratory protocols and own benefits . Embryos are cultured individually or in groups and the culturing conditions vary according to the lab. The culturing media are usually covered with a mineral well to prevent evaporation [89, 90]. Even though different media are developed and used, they can never fully imitate the culture environment of the oviduct. Also, the method of selecting the best culture media remains controversial. Embryo development depends on variable factors. Embryos in less media and a confined surface area showed better development than those in larger vessels, possibly because the secreted compounds are concentrated, as separately observed in the experiments performed by Thous et al. and Ali et al. [91, 92]. Also, Clark et al.  designed a microchannel in the microfluidic system to mimic the environment and function of the oviduct, which reduced the polyspermy and increased the potentially viable embryos. It has also been noted that the platform used for culture can influence the preimplantation embryo development .
According to various experiments performed on animals, it has also been observed that embryo development is also enhanced by the embryo density  and the different growth factors such as autocrine/paracrine are presented and secreted by the embryo itself . Nevertheless, it has also been proven that embryos in closer proximity rather than dispersed show improved development, also depending on the quality of the companion embryos [96, 97, 98]. While the benefits of these grouping and spacing effects in human embryo development have been shown, extended work still needs to be performed . However, the use of sequential media has overtaken the use of single step media in an uninterrupted manner. With the development in science and technology, microfluidics technology has proven to be an advantage in recent scientific studies relating to ART. Microfluidics can provide a microenvironment, dynamic fluid environment, and dynamic chemical environment. Raty et al. demonstrated that mouse embryos cultured in microchannels had a greater number of blastocysts with a lower percentage of degenerated embryos than those with controlled microdrops . However, the retrieval of the cultured embryos from the microchannels remains uncertain . In 2004, Gu et al. developed a microfluidic device with computer programming to regulate the fluid flow in the microchannel . In another study, for culturing embryos within a fluidic device, not only dynamic media flow but also co-culture was employed. In “womb-on-a-chip” microfluidics, Mizuno et al. were able to grow endometrial cells in a lower chamber and the embryos were cultured in the upper chamber separated by a thin membrane .
The microfluidic actuation created by the automated movement of Braille pins helped to develop embryos with automated fluid pumping and valving sequences ; this enabled the single embryo culture and the ability to select embryos with the highest implantation potential. The experiment also demonstrated the real-time monitoring of glucose consumption by blastocyst stage embryos in which UV light was used and the assays were performed separately from the embryo culture. Figure 4c shows a schematic design of a microfluidic device based on Braille display for cell culture and assay system.
Evaporation has been the critical problem while working with the sub-microliter volumes of fluids using PDMS, even in humidified cell culture media. This has become the barrier for mouse embryo and human endothelial cell growth and development. To address the problem, a PDMS–parylene hybrid was developed  which, by maintaining its property, reduces the problem of evaporation and osmolality shift. Adsorption and osmolality shift were seen when the PDMS was left uncoated (without parylene). It was also reported that, since the PDMS is softer than the polystyrene, it better supported the development of embryo and placental development .
Many experiments have been performed regarding the human embryo but due to the lack of convincing results, it is finding it difficult to make its way into the clinics. An experiment performed by Kieslinger et al. showed the in vitro development of a 4-day-old embryo that grew to the blastocyst stage in a static culture condition . However, a great deal of research still needs to be performed to achieve authentic results in this area for commercial use.
Cryopreservation is the process of preserving the cells, tissues, and any other biological materials at a very cold temperature, usually in liquid nitrogen or liquid nitrogen vapor for further use . Pregnancy using a cryopreserved human oocyte was first reported in 1986 . Since then, cryopreservation has become an important aspect of ART. However, the storage and viability of living cells are difficult using the simple freezing process. Due to climate change and other occupational and environmental hazards such as some medications, the women can become infertile. Likewise, the quality of sperm can also decline , which leads to the condition of oligospermia and azoospermia  leading to infertility and subfertility. In such cases, the cryopreserved oocytes, sperms, and embryos can be the only hope for having children . Not only human, but also endangered flora and fauna can benefit from this technology.
For women who are undergoing clinical treatment, the process of cryopreservation can provide a possibility of later pregnancy. In cancer patients, who are often at risk of ovarian failure, cryopreservation of the ovarian tissue is necessary for replantation to preserve fertility. Recently, a group of researchers in Korea were able to implant cryopreserved ovarian tissue into a patient with rectal cancer and it lead to the successful growth of a follicle from which the oocytes were retrieved . Unfortunately, the patient did not conceive, but this provided hope for people who are suffering from the same condition.
While a protocol is followed for the cryopreservation of any biological cells, the procedure of certain steps can vary among laboratories. The cryopreservation technique is basically categorized into three: programmable slow freezing, vitrification, and low CPA vitrification . In the programmable slow freezing process, most cells are frozen at 1 °C, which minimizes ice injury to support cell survival , since oocytes are prone to cell injury . In the vitrification process, a higher concentration of cryoprotective agents (CPAs) is used to prevent ice formation. Conversely, in the low CPA vitrification process, ultra-rapid cooling is used to suppress ice formation. All the processes involved, such as ultra-rapid cooling, thawing, CPA loading, and CPA unloading are difficult steps to complete, but with the introduction of technology such as microfluidics, this has become considerably easier.
During cryopreservation, the loading and unloading of CPA causes harmful anisotonic conditions . In an experiment conducted by Zou et al., a small amount of human spermatozoa was able to be preserved using a PDMS chip, without the need to use a cryoprotectant . Considering the damage caused during the loading and unloading of the CPA and the osmotic shock caused by this process, a microfluidic channel was introduced with diffusion and laminar flow which reduced the osmotic shock. This approach improved the post-thaw cell survivability by up to 25% compared to the conventional cryopreservation method . Further, a group of researchers proposed a new protocol with a microfluidic device to avoid potential osmotic and toxic damage to the oocytes . In this protocol, they achieved the loading of CPA in less than 15 min with less than 10% reduction of oocyte volume.
Cryopreservation of mature oocytes is a technique used to preserve the reproductive capacity, but the process uses temperatures as low as − 196 °C (liquid nitrogen), which can cause genetic drifts . Several drawbacks remain, including the use of a suitable temperature, a suitable type of CPA and also the correct amount and concentration of CPAs. A better understanding of the freezing and thawing process would improve the condition of the cryopreservation.
While microfluidic technology has made noticeable advances in the field of ART, limitations still remain. Even though two decades have passed since its invention, difficulties remain in its application in real-life IVF laboratories. While microfluidic technology has the capacity to revolutionize the field of ART, it needs to be more user friendly and provide more convincing results related to human embryos. Also, the new devices should be simplified to facilitate their use in ART laboratories.
On the other hand, the newly introduced 3D printing system for microfluidic devices can overcome the traditional method, if it broadens its material choices along with its resolution which will eventually reduce the rough surface properties seen in a 3D-printed microchannel.
This work was supported by a grant from the National Research Foundation (NRF) of Korea, funded by the Korean Government (MSIP) (No. 2014R1A5A2010008), (MOE)(No. 2014R1A1A2056425), (MSIT) (No. 2017R1A2B1011004). The work was also supported by the Commercialization Promotion Agency (COMPA) for R&D Outcomes, funded by the Ministry of Science and ICT (MSIT) (No. 2018K000287).
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