JMST Advances

, Volume 1, Issue 1–2, pp 1–11 | Cite as

Microfluidic technology for in vitro fertilization (IVF)

  • Seema Thapa
  • Yun Seok HeoEmail author


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.


Cryopreservation Embryos Gametes In vitro fertilization (IVF) Microfluidics 

1 Introduction

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 [1]. 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 [2]. 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 [3]. In the years between 2008 and 2010, of the 4.4 million assisted reproductive technology (ART) processes initiated, 1.1 million babies were born [4]. 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)

Infertility and low fertility rate have been subjects of concern in the past, to which factors related to male infertility contribute 50% [5]. The inability to bear children, by either a couple or a single person, is no longer a problem since the birth of the first IVF child in 1978. Now, the use of ART has enabled infertile or same-sex couples as well as those who are single to have their own children. IVF and ICSI like ART are the most widely used technologies, the development of which is still ongoing [5, 6]. IVF technique involves the fertilization of male sperm and female eggs outside the body. The process needs to go through several steps for it to be complete and the success of obtaining healthy embryos and offspring depends on the efficiency of each step of the process. After the patient’s health condition has been verified, the oocytes are retrieved and hormonally stimulated. After the collection and classification of the eggs, insemination takes place for which the semen sample is provided by the male partner. Inseminated oocytes are placed in an incubator overnight at an ideal temperature and pH. Fertilization is assessed the next day. Embryos are generally transferred 3 days after insemination, while some are transferred 5–6 days after insemination, at the blastocyst stage. The number of embryos to be transferred is determined by the age of the patient, the condition of the embryos, and other related factors. In some cases, when embryos have a thick membrane, assisted hatching is also performed. The pregnancy is tested after 2 weeks by evaluating beta human chorionic gonadotropin (b-hCG) or by other factors. In the developed world, over 4 million IVF babies have been born since 1978, which happens to be largest in the U.S. with 1 in 75 new births followed by Japan and France. ( [7]. Overall, this technique is still complex and extremely expensive, and is thus inaccessible to most people. Furthermore, complications regarding the technique still remain. Lately, ARTs have resulted in unsuccessful and multiple pregnancies. However, these have become less prominent with sequential culture media and single embryo transfer system [8] in addition with other technologies such as microfluidics (Fig. 1).
Fig. 1

Schematic diagram of the overall process of in vitro fertilization

IVF and ICSI are the earliest developed artificial insemination techniques [1]. 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 [9]. 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 [10]. The success rate of ICSI depends on the motility of fresh retrieved or thawed sperm and on the maturity of the sperm selected [11]. 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 [14]. 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.

2.1 Microfluidics

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 [17]. Since then, this technology has been used in different fields such as chemistry [18], molecular biology, and developmental biology [19], 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 [23]. Beside these fields, it is also used in forensic science [24]. 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 [27]. Also, a clean room was mandatory for its production, the sealing process was time consuming, and the yield was low [28]. The use of polymer instead of glass and silicon as a substrate for fabrication was introduced in the late 1990s [29]. 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 [32]. Nowadays, polymers are commonly used since they are simple, inexpensive, and readily disposable [33]. 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 [35]. 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 [36].

The fabrication process of a PDMS-based microfluidic device is relatively simple. The PDMS microchannel is fabricated using a simple soft lithography process in which the substrate is prepared by spin-coating and baking of a photoresist. The PDMS reagent is then directly cast on the master mask or master micro-mold followed by patterning [37]. SU-8 and standard microfluidics are generally used as the micro-molds in the soft lithography process with PDMS [38]. Casting is then performed after the patterning process. Casting is usually carried out by mixing the curing reagents at a 10:1 ratio followed by degassing and baking at 65 °C for 4 h. The PDMS is then peeled from the micro-mold to complete the process of casting. The PDMS is cut and the inlet and outlet holes are punched as required. The PDMS piece is then bonded with a glass slide or other material to complete the PDMS-based microfluidic device. The surface of the PDMS is treated with oxygen plasma, which forms a covalent bond between the silicon and oxygen which is a strong bond [39]. The overall procedure is summarized in Fig. 2.
Fig. 2

Schematic diagram of the fabrication process of the PDMS microchip

Pamela et al. demonstrated the applications of different types of polymers in microfluidic technology [40]. 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 [41]. 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 [44]. 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 [45].

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 [46]. 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 [47].

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 [48]. 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 [49]. 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 [52].

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 [53]. However, while the threat of oocyte lysis while inserting the needle into the oocyte still remains [54], the causes of oocyte degeneration have seldom been investigated [55]. One of the reasons for oocyte degeneration is technician error [56]. During the ICSI process, for fertilization, a single sperm is injected into the oocyte obtained after maturation [55]. Gonadotropin is important for controlled ovarian stimulation, as it helps to produce the optimum number of oocytes [57]. 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 [60]. 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 [60].

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 [48]. 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 [61]. ( These processes are known to enhance the efficiency of the process. However, centrifugation is said to cause sub-lethal damage to the sperm [62]. 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 [71].

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 [72]. 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 [73]. 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 [74]. Various microfluidic applications were later added that used electrical impedance and other technologies such as oriented sperm swimming technology to count the sperm [75]. 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 [81].

2.2.2 Sperm sorting

Sperm sorting is performed to select the best quality sperm to increase the fertilization and pregnancy rate during the process of ART. Conventional sperm preparation techniques such as swim up, density gradient centrifugation, and glass wool filtration are now obsolete, as they cannot produce the expected sperm populations for ART [71]. Many sperm sorting techniques have been developed with different unique functions. A method of separating sperm from epithelial cells was demonstrated using a micro-fabricated microfluidic device for potential application in cases of sexual assault [82]. In another experiment, motile sperm were separated from non-motile sperm using horizontally oriented gravity-driven sample inlet and outlet reservoirs [83]. Recently, Lin et al. developed a microfluidic device with a diffuser type chamber based on the speed of the sperm [84]. In the papers mentioned above, while the living/motile were sorted from the dead/motile, the concentrations of the motile sperm were not known. In an experiment conducted by McCormack et al. the concentrations of motile sperms were determined by fluorescently labeling the sperm using a micro-machine device [74]. Similarly, the simple microfluidic design which sorts the sperm on the basis of motility is shown in Fig. 3b.
Fig. 3

Uses of microfluidic device in the IVF process. a Schematic drawing of uses of microfluidic in different processes of IVF. b Simple design of microfluidic for sperm analysis [76] in which the sperm is sorted based on mobility. c Two-layer microfluidic device for oocyte maturation [77], where the upper layer has microchannels and the lower layer has microchambers. d In the microfluidic device for fertilization, the oocyte and embryos can be parked at a certain place, while the laminar flow transports the sperm toward the oocyte [78]. e Schematic diagram of droplet transport in dielectric platform for dynamic culture of individual mouse embryo [79]. f Microfluidic device used in the process of cryopreservation. (i) Loading of CPA using microfluidic (ii) freezing and thawing of cell (iii) unloading of CPA with microfluidic [80]

Microfluidics, on other hand is also used for oocyte maturation and fertilization along with the culture of embryo. Some of the devices used in the process of IVF are shown in Figs. 3 and 4.
Fig. 4

1 (a) Schematic design of sperm sorting microfluidic device. (b) Initial design of sperm sorter device made of PDMS. (c) Microfluidic sperm sorting device modified with polystyrene for compatibility in clinical use [113]. 2 (a) and (b) Overall system and a schematic diagram of Braille-based microfluidic embryo culture and metabolic assay system. Reprinted with authorized permission from Heo et al. [103]. (c) Schematic drawing of a micro-funnel culture device. (d) The micro-funnel where the embryos are loaded and cultured under the flow-through condition is created by the pin actuation sequence. Reprinted with authorized permission from Heo et al. [114]. 3 (a) The modified microfluidic device used for in vitro fertilization of mouse oocytes. The size of the device is the same as that of a U.S dime, where the microchannels are filled with blue dye and the microchannel gate system allows free flow of sperm and media [49]. (b) First microfluidic device for fertilization of pig oocyte [48]. 4 A two-layer PDMS microfluidic device with lower microfluidic network layer and upper control layer for handling a single oocyte for perfusion [115]

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 [85]. 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 [86]. 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 [24]. 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 [87]. Conventionally, gametes and embryos are cultured in different culturing dishes with different laboratory protocols and own benefits [88]. 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. [78] 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 [93].

According to various experiments performed on animals, it has also been observed that embryo development is also enhanced by the embryo density [94] and the different growth factors such as autocrine/paracrine are presented and secreted by the embryo itself [95]. 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 [99]. 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 [100]. However, the retrieval of the cultured embryos from the microchannels remains uncertain [54]. In 2004, Gu et al. developed a microfluidic device with computer programming to regulate the fluid flow in the microchannel [101]. 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 [102].

The microfluidic actuation created by the automated movement of Braille pins helped to develop embryos with automated fluid pumping and valving sequences [103]; 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 [104] 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 [105].

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 [106]. However, a great deal of research still needs to be performed to achieve authentic results in this area for commercial use.

2.2.4 Cryopreservation

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 [107]. Pregnancy using a cryopreserved human oocyte was first reported in 1986 [108]. 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 [109], which leads to the condition of oligospermia and azoospermia [110] leading to infertility and subfertility. In such cases, the cryopreserved oocytes, sperms, and embryos can be the only hope for having children [111]. 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 [112]. 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 [116]. In the programmable slow freezing process, most cells are frozen at 1 °C, which minimizes ice injury to support cell survival [117], since oocytes are prone to cell injury [118]. 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 [119]. 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 [120]. 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 [80]. Further, a group of researchers proposed a new protocol with a microfluidic device to avoid potential osmotic and toxic damage to the oocytes [115]. 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 [121]. 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.

3 Conclusions

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).


  1. 1.
    P.C. Steptoe, R.G. Edwards, Birth after the reimplantation of a human embryo. Lancet 2(8085), 366 (1978)Google Scholar
  2. 2.
    G. Palermo et al., Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet 340(8810), 17–18 (1992)Google Scholar
  3. 3.
    M. Bonduelle et al., Seven years of intracytoplasmic sperm injection and follow-up of 1987 subsequent children. Hum. Reprod. 14(suppl_1), 243–264 (1999)Google Scholar
  4. 4.
    S. Dyer et al., International committee for monitoring assisted reproductive technologies world report: assisted reproductive technology 2008, 2009 and 2010†. Hum. Reprod. 31(7), 1588–1609 (2016)Google Scholar
  5. 5.
    X. Zhang et al., Lensless imaging for simultaneous microfluidic sperm monitoring and sorting. Lab. Chip 11(15), 2535–2540 (2011)Google Scholar
  6. 6.
    The European, I.V.F.M.C. et al., Assisted reproductive technology in Europe, 2011: results generated from European registers by ESHRE†. Hum. Reprod. 31(2), 233–248 (2016)Google Scholar
  7. 7.
    B.C.J.M. Fauser, G.I. Serour, Introduction: optimal in vitro fertilization in 2020: the global perspective. Fertil. Steril. 100(2), 297–298 (2013)Google Scholar
  8. 8.
    D.K. Gardner, M. Lane, Towards a single embryo transfer. Reprod. BioMed. Online 6(4), 470–481 (2003)Google Scholar
  9. 9.
    S.E. Lanzendorf et al., A preclinical evaluation of pronuclear formation by microinjection of human spermatozoa into human oocytes. Fertil. Steril. 49(5), 835–842 (1988)Google Scholar
  10. 10.
    A. Van Steirteghem, Celebrating ICSI’s twentieth anniversary and the birth of more than 2.5 million children—the ‘how, why, when and where’. Hum. Reprod. 27(1), 1–2 (2012)Google Scholar
  11. 11.
    A.R. Ergur et al., Sperm maturity and treatment choice of in vitro fertilization (IVF) or intracytoplasmic sperm injection: diminished sperm HspA2 chaperone levels predict IVF failure. Fertil. Steril. 77(5), 910–918 (2002)Google Scholar
  12. 12.
    Y.-S. Park et al., Influence of motility on the outcome of in vitro fertilization/intracytoplasmic sperm injection with fresh vs. frozen testicular sperm from men with obstructive azoospermia. Fert. Steril. 80(3), 526–530 (2003)Google Scholar
  13. 13.
    H. Shibahara et al., Correlation between the motility of frozen-thawed epididymal spermatozoa and the outcome of intracytoplasmic sperm injection. Int. J. Androl. 22(5), 324–328 (1999)Google Scholar
  14. 14.
    E.D. Kim, An overview of male infertility in the era of intracytoplasmic sperm injection. Zhonghua Yi Xue Za Zhi (Taipei) 64(2), 71–83 (2001)Google Scholar
  15. 15.
    W. Verpoest, H. Tournaye, ICSI: hype or hazard? Hum. Fertil. (Camb) 9(2), 81–92 (2006)Google Scholar
  16. 16.
    P. Devroey, A. Van Steirteghem, A review of ten years experience of ICSI. Hum. Reprod. Update 10(1), 19–28 (2004)Google Scholar
  17. 17.
    S.C. Terry, J.H. Jerman, J.B. Angell, A gas chromatographic air analyzer fabricated on a silicon wafer. IEEE Trans. Electron Devices 26(12), 1880–1886 (1979)Google Scholar
  18. 18.
    J. Abramczuk, D. Solter, H. Koprowski, The beneficial effect EDTA on development of mouse one-cell embryos in chemically defined medium. Dev. Biol. 61(2), 378–383 (1977)Google Scholar
  19. 19.
    D. Beebe et al., Microfluidic technology for assisted reproduction. Theriogenology 57(1), 125–135 (2002)Google Scholar
  20. 20.
    A.J. Tomlinson, N.A. Guzman, S. Naylor, Enhancement of concentration limits of detection in CE and CE-MS: a review of on-line sample extraction, cleanup, analyte preconcentration, and microreactor technology. J. Capill. Electrophor. 2(6), 247–266 (1995)Google Scholar
  21. 21.
    J.P. Brody, P. Yager, Diffusion-based extraction in a microfabricated device. Sens. Actuators A 58(1), 13–18 (1997)Google Scholar
  22. 22.
    R.L. Krisher, M.B. Wheeler, Towards the use of microfluidics for individual embryo culture. Reprod. Fertil. Dev. 22(1), 32–39 (2009)Google Scholar
  23. 23.
    C. Yi et al., Microfluidics technology for manipulation and analysis of biological cells. Anal. Chim. Acta 560(1), 1–23 (2006)MathSciNetGoogle Scholar
  24. 24.
    V.F.S. Tsai et al., Application of microfluidic technologies to the quantification and manipulation of sperm. Urol. Sci. 27(2), 56–59 (2016)Google Scholar
  25. 25.
    P.C.H. Li, D.J. Harrison, Transport, manipulation, and reaction of biological cells on-chip using electrokinetic effects. Anal. Chem. 69(8), 1564–1568 (1997)Google Scholar
  26. 26.
    O. Fumihiro, N. Yuta, I. Takanori, Measurement of the electrophoretic mobility of sheep erythrocytes using microcapillary chips. Electrophoresis 26(6), 1163–1167 (2005)Google Scholar
  27. 27.
    T.D. Boone et al., Plastic advances microfluidic devices. Anal. Chem. 74(3), 78a–86a (2002)Google Scholar
  28. 28.
    J.C. McDonald, G.M. Whitesides, Poly(dimethylsiloxane) as a material for fabricating microfluidic devices. Acc. Chem. Res. 35(7), 491–499 (2002)Google Scholar
  29. 29.
    A. Manz, N. Graber, H.M. Widmer, Miniaturized total chemical analysis systems: a novel concept for chemical sensing. Sens. Actuators B Chem. 1(1), 244–248 (1990)Google Scholar
  30. 30.
    A. Alrifaiy, O.A. Lindahl, K. Ramser, Polymer-based microfluidic devices for pharmacy, biology and tissue engineering. Polymers 4(3), 1349 (2012)Google Scholar
  31. 31.
    P.M. van Midwoud et al., Comparison of biocompatibility and adsorption properties of different plastics for advanced microfluidic cell and tissue culture models. Anal. Chem. 84(9), 3938–3944 (2012)Google Scholar
  32. 32.
    L. Martynova et al., Fabrication of plastic microfluid channels by imprinting methods. Anal. Chem. 69(23), 4783–4789 (1997)Google Scholar
  33. 33.
    H. Becker, L.E. Locascio, Polymer microfluidic devices. Talanta 56(2), 267–287 (2002)Google Scholar
  34. 34.
    S.K. Sia, G.M. Whitesides, Microfluidic devices fabricated in Poly(dimethylsiloxane) for biological studies. Electrophoresis 24(21), 3563–3576 (2003)Google Scholar
  35. 35.
    M.B. Wheeler, E.M. Walters, D.J. Beebe, Toward culture of single gametes: the development of microfluidic platforms for assisted reproduction. Theriogenology 68, S178–S189 (2007)Google Scholar
  36. 36.
    E. Berthier, E.W. Young, D. Beebe, Engineers are from PDMS-land, Biologists are from Polystyrenia. Lab Chip 12(7), 1224–1237 (2012)Google Scholar
  37. 37.
    Y. Xia, G.M. Whitesides, Soft lithography. Ann. Rev. Mater. Sci. 28(1), 153–184 (1998)Google Scholar
  38. 38.
    M.A. Unger et al., Monolithic microfabricated valves and pumps by multilayer soft lithography. Science 288(5463), 113–116 (2000)Google Scholar
  39. 39.
    Z.Z. Chong et al., Acoustofluidic control of bubble size in microfluidic flow-focusing configuration. Lab. Chip 15(4), 996–999 (2015)Google Scholar
  40. 40.
    P.N. Nge, C.I. Rogers, A.T. Woolley, Advances in microfluidic materials, functions, integration, and applications. Chem. Rev. 113(4), 2550–2583 (2013)Google Scholar
  41. 41.
    E. Sollier et al., Rapid prototyping polymers for microfluidic devices and high pressure injections. Lab. Chip 11(22), 3752–3765 (2011)Google Scholar
  42. 42.
    J.-Y. Kim et al., Thermoset polyester droplet-based microfluidic devices for high frequency generation. Lab. Chip 11(23), 4108–4112 (2011)Google Scholar
  43. 43.
    G.S. Fiorini et al., Fabrication improvements for thermoset polyester (TPE) microfluidic devices. Lab. Chip 7(7), 923–926 (2007)Google Scholar
  44. 44.
    W. Li et al., Microfluidic fabrication of microparticles for biomedical applications. Chem. Soc. Rev. 47(15), 5646–5683 (2018)Google Scholar
  45. 45.
    H.-Y. Huang, Y.-L. Lai, D.-J. Yao, Dielectrophoretic microfluidic device for in vitro fertilization. Micromachines 9, 135 (2018)Google Scholar
  46. 46.
    S. Waheed et al., 3D printed microfluidic devices: enablers and barriers. Lab. Chip 16(11), 1993–2013 (2016)Google Scholar
  47. 47.
    C. Chen et al., 3D-printed microfluidic devices: fabrication, advantages and limitations-a mini review. Anal. Methods 8(31), 6005–6012 (2016)Google Scholar
  48. 48.
    J.E. Swain et al., Thinking big by thinking small: application of microfluidic technology to improve ART. Lab. Chip 13(7), 1213–1224 (2013)Google Scholar
  49. 49.
    R.S. Suh et al., IVF within microfluidic channels requires lower total numbers and lower concentrations of sperm. Hum. Reprod. 21(2), 477–483 (2006)MathSciNetGoogle Scholar
  50. 50.
    H.C. Zeringue, J.J. Rutledge, D.J. Beebe, Early mammalian embryo development depends on cumulus removal technique. Lab. Chip 5(1), 86–90 (2005)Google Scholar
  51. 51.
    H.C. Zeringue, D.J. Beebe, M.B. Wheeler, Removal of cumulus from mammalian zygotes using microfluidic techniques. Biomed. Microdevice 3(3), 219–224 (2001)Google Scholar
  52. 52.
    H.C. Zeringue, M.B. Wheeler, D.J. Beebe, A microfluidic method for removal of the zona pellucida from mammalian embryos. Lab. Chip 5(1), 108–110 (2005)Google Scholar
  53. 53.
    A.C. Van Steirteghem et al., High fertilization and implantation rates after intracytoplasmic sperm injection. Hum. Reprod. 8(7), 1061–1066 (1993)Google Scholar
  54. 54.
    G.D. Smith, S. Takayama, Application of microfluidic technologies to human assisted reproduction. Mol. Hum. Reprod. 23(4), 257–268 (2017)Google Scholar
  55. 55.
    M.P. Rosen et al., Oocyte degeneration after intracytoplasmic sperm injection: a multivariate analysis to assess its importance as a laboratory or clinical marker. Fertil. Steril. 85(6), 1736–1743 (2006)Google Scholar
  56. 56.
    J.C.M. Dumoulin et al., Embryo development and chromosomal anomalies after ICSI: effect of the injection procedure*. Hum. Reprod. 16(2), 306–312 (2001)Google Scholar
  57. 57.
    R. Howie, V. Kay, Controlled ovarian stimulation for in vitro fertilization. Br. J. Hosp. Med. (London, England 2005) 79(4), 194–199 (2018)Google Scholar
  58. 58.
    S. Bhattacharya et al., Conventional in vitro fertilisation versus intracytoplasmic sperm injection for the treatment of non-male-factor infertility: a randomised controlled trial. The Lancet 357(9274), 2075–2079 (2001)Google Scholar
  59. 59.
    M. Eftekhar et al., Comparison of conventional IVF versus ICSI in non-male factor, normoresponder patients. Iranian J Reprod Med 10(2), 131–136 (2012)Google Scholar
  60. 60.
    K. Matsuura et al., A microfluidic device to reduce treatment time of intracytoplasmic sperm injection. Fertil. Steril. 99(2), 400–407 (2013)Google Scholar
  61. 61.
    World Health Organization, WHO laboratory manual for the examination and processing of human semen, 5th edn. (World Health Organization, Geneva, 2010)Google Scholar
  62. 62.
    J.G. Alvarez et al., Centrifugation of human spermatozoa induces sublethal damage; separation of human spermatozoa from seminal plasma by a dextran swim-up procedure without centrifugation extends their motile lifetime. Hum. Reprod. 8(7), 1087–1092 (1993)Google Scholar
  63. 63.
    G. Barroso, M. Morshedi, S. Oehninger, Analysis of DNA fragmentation, plasma membrane translocation of phosphatidylserine and oxidative stress in human spermatozoa. Hum. Reprod. 15(6), 1338–1344 (2000)Google Scholar
  64. 64.
    A.R. John, J.S. Clarkson, Significance of reactive oxygen species and antioxidants in defining the efficacy of sperm preparation techniques. J. Androl. 9(6), 367–376 (1988)Google Scholar
  65. 65.
    C.M. Hughes et al., The effects of antioxidant supplementation during Percoll preparation on human sperm DNA integrity. Hum. Reprod. 13(5), 1240–1247 (1998)Google Scholar
  66. 66.
    D.G. Pyne et al., Digital microfluidic processing of mammalian embryos for vitrification. PLoS ONE 9(9), e108128 (2014)Google Scholar
  67. 67.
    M. Benchaib et al., Sperm DNA fragmentation decreases the pregnancy rate in an assisted reproductive technique. Hum. Reprod. 18(5), 1023–1028 (2003)Google Scholar
  68. 68.
    F.N. Wang et al., Modification of the wang tube to improve in vitro semen manipulation. Arch. Androl. 29(3), 267–269 (1992)MathSciNetGoogle Scholar
  69. 69.
    C.H. Lih et al., Development of a microchamber which spontaneously selects high-quality sperm for use in in vitro fertilization or micromanipulation. J. Assist. Reprod. Genet. 13(8), 657–662 (1996)Google Scholar
  70. 70.
    F.N. Wang, Realtime sperm separation system: a review of wang tubes and related technologies. Arch. Androl. 34(1), 13–32 (1995)Google Scholar
  71. 71.
    R. Henkel, Sperm preparation: state-of-the-art—physiological aspects and application of advanced sperm preparation methods. Asian J. Androl. 14(2), 260–269 (2012)Google Scholar
  72. 72.
    R.T. Schulte et al., Microfluidic sperm sorting device provides a novel method for selecting motile sperm with higher DNA integrity. Fertil. Steril. 88, S76 (2007)Google Scholar
  73. 73.
    R. Nosrati et al., Rapid selection of sperm with high DNA integrity. Lab. Chip 14(6), 1142–1150 (2014)Google Scholar
  74. 74.
    M.C. McCormack, S. McCallum, B. Behr, A novel microfluidic device for male subfertility screening. J. Urol. 175(6), 2223–2227 (2006)Google Scholar
  75. 75.
    L.I. Segerink et al., On-chip determination of spermatozoa concentration using electrical impedance measurements. Lab. Chip 10(8), 1018–1024 (2010)Google Scholar
  76. 76.
    H.Y. Huang, H.T. Fu, H.Y. Tsing, H.J. Huang, C.J. Li et al., Motile human sperm sorting by an integrated microfluidic system. J. Nanomed. Nanotechnol. 5, 199 (2014). Google Scholar
  77. 77.
    S. Zargari et al., A microfluidic chip for in vitro oocyte maturation. Sens. Lett. 14, 435–440 (2015)Google Scholar
  78. 78.
    S.G. Clark et al., Reduction of polyspermic penetration using biomimetic microfluidic technology during in vitro fertilization. Lab. Chip 5(11), 1229–1232 (2005)Google Scholar
  79. 79.
    H.Y. Huang et al., Digital microfluidic dynamic culture of mammalian embryos on an electrowetting on dielectric (EWOD) chip. PLoS ONE 10(5), e0124196 (2015)Google Scholar
  80. 80.
    Y.S. Song et al., Microfluidics for cryopreservation. Lab. Chip 9(13), 1874–1881 (2009)Google Scholar
  81. 81.
    C. Ainsworth, B. Nixon, R.J. Aitken, Development of a novel electrophoretic system for the isolation of human spermatozoa. Hum. Reprod. 20(8), 2261–2270 (2005)Google Scholar
  82. 82.
    K.M. Horsman et al., separation of sperm and epithelial cells in a microfabricated device: potential application to forensic analysis of sexual assault evidence. Anal. Chem. 77(3), 742–749 (2005)Google Scholar
  83. 83.
    B. Cho, et al. A microfluidic device for separating motile sperm from nonmotile sperm via inter-streamline crossings. in Proceedings (Cat. No.02EX578) od 2nd annual international IEEE-EMBS special topic conference on microtechnologies in medicine and biology; 2002Google Scholar
  84. 84.
    L. Yu-Nan, et al. Micro diffuser-type movement inversion sorter for high-efficient sperm sorting. in The 8th annual IEEE international conference on nano/micro engineered and molecular systems; 2013Google Scholar
  85. 85.
    H.Y. Huang, H.T. Fu, H.Y. Tsing, H.J. Huang, C.J. Li, Motile human sperm sorting by an integrated microfluidic system. Nanomed Nanotechnol 5, 2 (2014)Google Scholar
  86. 86.
    B. Shao et al., in automated motile cell capture and analysis with optical traps, Methods in cell biology. 2007, Academic Press. p. 601–627Google Scholar
  87. 87.
    D.K. Gardner, M. Lane, Culture of viable human blastocysts in defined sequential serum-free media. Hum. Reprod. 13(suppl_3), 148–159 (1998)Google Scholar
  88. 88.
    J.E. Swain, G.D. Smith, Advances in embryo culture platforms: novel approaches to improve preimplantation embryo development through modifications of the microenvironment. Hum. Reprod. Update 17(4), 541–557 (2011)Google Scholar
  89. 89.
    B. Balaban, D. Sakkas, D.K. Gardner, Laboratory procedures for human in vitro fertilization. Semin Reprod. Med. 32(4), 272–282 (2014)Google Scholar
  90. 90.
    J.E. Swain, Optimal human embryo culture. Semin Reprod. Med. 33(2), 103–117 (2015)MathSciNetGoogle Scholar
  91. 91.
    G. Thouas, G. Jones, A. Trounson, The ‘GO’ system—a novel method of microculture for in vitro development of mouse zygotes to the blastocyst stage. Reproduction 126(2), 161–169 (2003)Google Scholar
  92. 92.
    J. Ali, Continuous Ultra Micro-Drop (cUMD) Culture yields higher pregnancy and implantation rates than either large-drop culture or fresh medium replacement. Clin Embryol 7, 17–23 (2004)Google Scholar
  93. 93.
    G.D. Smith, S. Takayama, J.E. Swain, Rethinking in vitro embryo culture: new developments in culture platforms and potential to improve assisted reproductive technologies. Biol. Reprod. 86(3), 62 (2012)Google Scholar
  94. 94.
    M.G. Katz-Jaffe, W.B. Schoolcraft, D.K. Gardner, Analysis of protein expression (secretome) by human and mouse preimplantation embryos. Fertil. Steril. 86(3), 678–685 (2006)Google Scholar
  95. 95.
    K.S. Richter, The importance of growth factors for preimplantation embryo development and in vitro culture. Curr. Opin. Obstet. Gynecol. 20(3), 292–304 (2008)Google Scholar
  96. 96.
    P.J. Stokes, L.R. Abeydeera, H.J. Leese, Development of porcine embryos in vivo and in vitro; evidence for embryo ‘cross talk’ in vitro. Dev. Biol. 284(1), 62–71 (2005)Google Scholar
  97. 97.
    T. Somfai et al., Culture of bovine embryos in polyester mesh sections: the effect of pore size and oxygen tension on in vitro development. Reprod. Domes. Anim. 45(6), 1104–1109 (2010)Google Scholar
  98. 98.
    R.E. Spindler et al., Improved felid embryo development by group culture is maintained with heterospecific companions. Theriogenology 66(1), 82–92 (2006)MathSciNetGoogle Scholar
  99. 99.
    M. Almagor et al., Pregnancy rates after communal growth of preimplantation human embryos in vitro *. Fertil. Steril. 66(3), 394–397 (1996)Google Scholar
  100. 100.
    S. Raty et al., Embryonic development in the mouse is enhanced via microchannel culture. Lab Chip 4(3), 186–190 (2004)Google Scholar
  101. 101.
    W. Gu et al., Computerized microfluidic cell culture using elastomeric channels and Braille displays. Proc. Natl. Acad. Sci. USA 101(45), 15861–15866 (2004)Google Scholar
  102. 102.
    J. Mizuno et al., Development of microfluidic embryo co-culture system for human ART. Fertil. Steril. 84, S403 (2005)Google Scholar
  103. 103.
    Y.S. Heo et al., Real time culture and analysis of embryo metabolism using a microfluidic device with deformation based actuation. Lab. Chip 12(12), 2240–2246 (2012)Google Scholar
  104. 104.
    Y.S. Heo et al., Characterization and resolution of evaporation-mediated osmolality shifts that constrain microfluidic cell culture in poly(dimethylsiloxane) devices. Anal. Chem. 79(3), 1126–1134 (2007)Google Scholar
  105. 105.
    K.S. Kolahi et al., Effect of substrate stiffness on early mouse embryo development. PLoS ONE 7(7), e41717 (2012)Google Scholar
  106. 106.
    D.C. Kieslinger et al., In vitro development of donated frozen-thawed human embryos in a prototype static microfluidic device: a randomized controlled trial. Fertil. Steril. 103(3), 680-6.e2 (2015)Google Scholar
  107. 107.
    L. Kuleshova, D. Hutmacher, Chapter 13cryobiology, in Tissue engineering. (Academic Press, Burlington, 2008). p. 363–401Google Scholar
  108. 108.
    C. Chen, Pregnancy after human oocyte cryopreservation. Lancet 1(8486), 884–886 (1986)Google Scholar
  109. 109.
    H. Merzenich, H. Zeeb, M. Blettner, Decreasing sperm quality: a global problem? BMC Public Health 10(1), 24 (2010)Google Scholar
  110. 110.
    ESHRE Capri Workshop Group, Europe the continent with the lowest fertility. Hum Reprod Update 16(6), 590–602 (2010)Google Scholar
  111. 111.
    J.K. Choi, H. Huang, X. He, Improved low-CPA vitrification of mouse oocytes using quartz microcapillary. Cryobiology 70(3), 269–272 (2015)Google Scholar
  112. 112.
    J.R. Lee et al., Successful in vitro fertilization and embryo transfer after transplantation of cryopreserved ovarian tissue: report of the first korean case. J. Korean Med. Sci. 33(21), e156 (2018)Google Scholar
  113. 113.
    T.G. Schuster et al., Isolation of motile spermatozoa from semen samples using microfluidics. Reprod. BioMed. Online 7(1), 75–81 (2003)Google Scholar
  114. 114.
    Y.S. Heo et al., Dynamic microfunnel culture enhances mouse embryo development and pregnancy rates. Hum. Reprod. 25(3), 613–622 (2010)Google Scholar
  115. 115.
    Y.S. Heo et al., Controlled loading of cryoprotectants (CPAs) to oocyte with linear and complex CPA profiles on a microfluidic platform. Lab. Chip 11(20), 3530–3537 (2011)Google Scholar
  116. 116.
    G. Zhao, J. Fu, Microfluidics for cryopreservation. Biotechnol. Adv. 35(2), 323–336 (2017)Google Scholar
  117. 117.
    P. Mazur, Freezing of living cells: mechanisms and implications. Am. J. Physiol. Cell Physiol. 247(3), C125–C142 (1984)Google Scholar
  118. 118.
    Y. Zeron et al., Kinetic and temporal factors influence chilling injury to germinal vesicle and mature bovine oocytes. Cryobiology 38(1), 35–42 (1999)MathSciNetGoogle Scholar
  119. 119.
    N. Ogonuki et al., Spermatozoa and spermatids retrieved from frozen reproductive organs or frozen whole bodies of male mice can produce normal offspring. Proc. Natl. Acad. Sci. USA 103(35), 13098–13103 (2006)Google Scholar
  120. 120.
    Y. Zou, T. Yin, S. Chen, J. Yang, W. Huang, On-chip cryopreservation: a novel method for ultra-rapid cryoprotectant- free cryopreservation of small amounts of human spermatozoa. PLoS ONE 8(4), e61593 (2013)Google Scholar
  121. 121.
    T.H. Jang et al., Cryopreservation and its clinical applications. Integr. Med. Res. 6(1), 12–18 (2017)Google Scholar

Copyright information

© The Korean Society of Mechanical Engineers 2019

Authors and Affiliations

  1. 1.Department of Biomedical Engineering, School of MedicineKeimyung UniversityDaeguSouth Korea

Personalised recommendations