Numerous experiences have now been available as a result of the development of Micro Electro Mechanical System (MEMS) technologies in medical applications, which may enable them to be used in a microfluidic system [1]. Controlling fluid movement is useful for cell analysis systems, medication delivery systems and assisted reproductive technologies; hence microfluidics has been widely used in biomedical sectors [2-3]. Microfluidic technology has been used in a variety of biological applications, mostly to miniaturize and simplify laboratory procedures [4]. In the field of medicine, reproductive medicine has played a significant role. The abnormalities and insufficient amount of spermatozoa are the main causes of male infertility. Assisted Reproductive Technologies (ART's) may be used to address these issues, with In Vitro Fertilization (IVF) and Intracytoplasmic Sperm Injection (ICSI) being two common options [5]. Traditional or conventional sperm sorting methods, such as sperm washing, which is a simple way to select live sperms by centrifugation once or twice, swim-up techniques, which is the most common method used in IVF procedure, in which motile spermatozoa were separated by their ability to swim out of the semen [6], Density Gradient Centrifugation (DGC), in which motile spermatozoa were selected by a density discontinuous gradient and sperm migration, which is Furthermore, these methods increase the likelihood of DNA damage [7]. Horsman et al. [8] used a microfabricated device based on the ability of motile spermatozoa to move out of their initial streamlines in the laminar fluid stream, while Cho et al. [9] proposed a horizontally-setup gravity-driven pumping system, which greatly simplifies the sorting step without the use of centrifugation and the purity of motile spermatozoa after separation was nearly 100%. 

In recent years, some novel sperm separation techniques have been developed. Lin et al. [10] presented a microfluidic system with a diffuser chamber that uses a velocity gradient to separate the spermatozoa. Ainsworth et al. [11] proposed a quick, safe and low-DNA-damage electrophoretic technique for isolating human spermatozoa based on size and electric field. The surface hydrophilicity of the channels and reservoirs was improved by coating them with 1% Bovine Serum Albumin (BSA) [12].

Poly (dimethylsiloxane) (PDMS) microfluidic devices have the potential to improve biological and medicinal capabilities. Wu et al. [13] described a PDMS-based sperm sorting device with a PEG-MA-coated surface that is moderately hydrophilic and non-fouling. The microfluidic device can successfully separate raw semen samples into immotile and motile groups without centrifugation steps, as demonstrated in an earlier study [14]. Simultaneously, the sperms' harm might be avoided. In addition, the microfluidic device can shorten the time it takes to perform other ART's, which is utilized to boost sperm concentration in low-quality sperm samples [15]. The findings show that this device could be used in human ART's for ICSI treatment. However, no method was found in the aforementioned research to corroborate the sperm quantity test, which can quickly analyze motility data. The amount of spermatozoa contained living and dead sperm may be defined using fluorescent stain [16] and statistics methods such as flow cytometric analysis [17] and a statistic model for measuring the sorting efficiency of motile sperms was created. The concept of isolating mouse spermatozoa using a microfluidic technology has been demonstrated [18].

In this study, the integrated microfluidic system which consists of laminar stream-based microchannels demonstrated the separation of human motile spermatozoa and flow cytometric analysis was used to enhance the sperm motility sorting efficiency (Figure 1). Considering the characteristic of the microfluidic channel, it is similar to the natural environment of the fallopian. The interactions between laminar flow and sperm activity were tested as well. Due to the laminar flow in different directions, the semen sample was loaded into a straight laminar flow and the results showed that most motile spermatozoa with lateral activity could swim across the two fluid boundaries to be sorted (Figure 2). The working concept lies in the design of multiple channel systems that control the formation of multiple laminar streams and motile spermatozoa could swim across the stream lines and were collected by the dedicated reservoirs. 

Figure 1: Schematic Illustration of Sorting Process for Microfluidic System

Figure 2: The Schematics of Multiple Channel Microfluidic Chips. The Motile Sperms would be Sorted Either with Random Swimming Direction by Laminar Flow                                                                       

Microscopic examination revealed that spermatozoa with different motility. Moreover, Microfluidic platforms for sperm sorting rely on either active or passive methods. In active methods, external stimulators, such as the temperature of chemical gradients or an active fluid flow, perform the sorting, while passive methods rely on the inherent behavior and movement of spermatozoa in the absence of any external stimuli [19].

A Total of 30 Asthenospermic males were involved in this study during their attendance to the infertility clinic at High Institute for Infertility Diagnosis and Assisted Reproductive Technologies; Al- Nahrain University and AL-Farah Specialist Fertility Center. The collection of semen samples and seminal fluid analysis was assessed or done accordingly to WHO.

Each semen sample was divided into 4 parts. The first part was prepared for sperm characterization and assessment of before activation, the second part was used after activation by Centrifugation technique (Indirect Swim Up) (Figure 3), the third part was used after activation by Simple layer (Direct Swim Up) (Figure 4), while the last part was used after activation by Microfluidic Chamber (Figure 5).

Fertile Microfluidic Sperm Sorting Chip or Microfluidic Chamber Principle

Chemical free method of sorting sperm using a disposable chip, the method is based on the principle of natural sperm selection in a passage through micro-barriers imitating natural environment of female reproductive system (fallopian tubes). 

Design

Fertile Plus: is designed for sorting sperms which have better morphology, genetic and physiologic quality from other dead or immature sperms without using sperm techniques, like centrifuge or mixing with pipette, in every assisted reproductive technology andrology or embryology laboratory.

Fertile Plus has an inlet, for applying the sample and an outlet for collecting processed product. Fertile Plus represented sterile and single-use to users. Also, product mimics female reproducing system with micro-fluiding technology for natural sperm movement.

Fertile Plus manufactured with biocompatible materials. It is designed for to have best ergonomic working safety and user friendliness.

Microfluidic Chamber is a new gentle alternative to standard sperm processing which does not require any previous processing of sperm sample or use of devices (e.g. centrifuge) that increase oxidative stress in sperm.

How Does the Method Work?

The chip or chamber has an inlet for the sample, which is linked to the outlet opening with a microfluidic duct. The untreated sperm sample is pipetted into the inlet where the present sperm must actively migrate in the outlet against the movement of the fluid as well as through the above-mentioned micro-barriers. Sperm cells are sorted by separating healthy motile sperm in the outlet from poor-quality sperm captured in the duct (Figure 5).

Figure 3: Showed Sperm Activation by Centrifugation (Indirect Swim up)

Figure 4: Showed Sperm Activation by Simple Layer (Direct Swim up)

Figure 5: Showed Sperm Activation by (FERTILE Plus) Microfluidic Chamber (MFC)

Performance Evaluation

The efficiency of a sperm preparation or selection technique is usually and practically expressed as:

Concentration

After working with FERTILE Plus, collected spermatozoa will be compared to unprocessed semen and other sperm preparation techniques.

Motility

After working with FERTILE Plus, collected sperms has more than 2 times motility compared to unprocessed semen.

Sperm Morphologic Analysis

Higher percentage of sperm sorted using FERTILE Plus chips were morphologically normal than stock sperm and other sperm preparation techniques.

Statistical Analysis

The Statistical Analysis System-SAS (2012) program was used to detect the effect of difference factors in study parameters. Least Significant Difference (LSD) test Analysis of Variation (ANOVA) was used to significant compare between means in this study.

By Assessment of different parameters of semen analysis in the three sperm preparation techniques, it found that; the Third Technique (MFC) had good parameters in relation to the other techniques and it had a significant difference in relation to the Morphologically Normal Sperm (MNS) of the sperm parameters as follows:

In before activation, the result was (26.67±1.02 c), in the activation technique by centrifugation, the result was (56.33±1.52 b), in the activation technique by simple layer, the result was (54.67±1.44 b) and lastly in the activation technique by Microfluidic Chamber, the result was (60.83±1.73 a).The results can be illustrated in Table 1 and Figure 6.

Results of ART's still do not exceed high percentages due to different factors, one of them is male factor infertility. Therefore the global companies concern on the techniques that used for activation of semen samples of infertile men. However, till now, there are many semen samples did not response to sperm preparation techniques leading to failure of sperm activation [20].

New technological advancements in the field have altered current infertility management. Microfluidics is one of them, as it mimics the normal biophysical and biochemical environment and allows for more physiological sperm selection in ART's patients. This approach is becoming more widely used in clinical practice. This study found some differences between MFC and the other techniques (conventional techniques), particularly in the Morphologically Normal Sperm parameter, which was significantly higher in the MFC. 

The primary outcome of this study is to find the best technique to be used in asthenozoospermic patients as a mimic to increase the fertilization rate and embryo quality. There were no previous studies or articles that made this wide comparison that relevant to the sperm preparation techniques superiority. 

Few studies have investigated new-generation sperm selection techniques using embryo quality. Gianaroli et al. suggested that sperm selected with an inverted microscope produced higher embryo quality and ongoing pregnancy rates compared with classical ICSI, but fertilization rates were similar [21]. In contrast, Balaban et al. [22] found no difference in the quality of embryos or live birth rates for IVF treatment using Intracytoplasmic Morphologically Selected Sperm Injection (IMSI) compared with classical ICSI [23]. In the meta-analysis of Magnetic-Activated Cell Sorting (MACS) (another new-generation sperm selection method) compared with classical ICSI, authors suggested that the pregnancy rate was higher in the MACS group (RR = 1.50, 95% CI 1.14–1.98). However, this study did not include data about the quality of embryos or the fertilization rate [24].

Table 1: Comparison among before Activation and Three Sperm Preparation Techniques in Different Study Parameters

Means having with the different letters in same column differed significantly. ** (p≤0.01), Conc. = Sperm Concentration (million/mL) GA,GB,GC,GD = Sperm Motility Grade%, MNS = Morphologically Normal Sperm%.

Figure 6: Comparison between Before Activation and Three Different Techniques in Sperm Concentration, Motility Grades.: GA, GB, GC, GD and Morphologically Normal Sperm 

Some studies report that gradient method was found to be significantly superior in selecting sperms with well-constructed morphology as well as in terms of ongoing pregnancy rates compared with the swim-up method [25-27].

It did not find significant difference in sperm parameters between the MFC technique and the other conventional techniques used except for the morphology parameter on which, there was a significant difference between MFC technique and the other techniques. Finally the sample size used in this study is usually determined and based on the cost of the Microfluidic Chamber and from the results obtained through this study it has been revealed the need for most suitable and effective sperm treatment in the laboratory of ART's.

Compared with the conventional sperm preparation or activation techniques, the developed Microfluidic Chamber with multiple channels improves the sorting efficiency in the quality and quantity of sperm.

Conflict of Interest

The author declared no potential conflicts of interest with respect to the research, authorship and/or publication of this article.

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