Utilization of a Fertile Chip in Cases of Male Infertility

Abstract

Infertility is a significant reproductive health issue affecting 10–15% of couples of reproductive age worldwide. The male component adds 30–50% to IVF failure. In the examination of male infertility, sperm count, morphology, motility, and genomic integrity of sperm are crucial factors. Several strategies for generating morphologically and genetically superior sperms for use in IUI and IVF procedures or experimental research have been developed. Density gradient and swim-up approaches are two of the most commonly used applications. As this procedure needs centrifugation, it has been observed that it may have a negative impact on sperm viability, increase oxygen radicals, and result in sperm DNA fragmentation. Inadequacies in sperm extraction procedures may have unfavorable long-term consequences in terms of fertilization success, continuation of pregnancy, and embryo health. Microfluidic sperm preparation is an alternate method for decreasing DNA fragmentation at this stage, despite the fact that it has only been established recently. However, these innovative techniques have little clinical trials. According to studies, sperm sorting chips are user-friendly, inexpensive, and do not require many manual stages.

1. Introduction

Infertility affects 10–15% of couples worldwide [1]. The malefactor of infertility is a cause of infertility in 40–50% of infertile couples, and it coexists with female infertility [2, 3]. One of the common causes of male infertility is low sperm count owing to primary testicular failure. Nutritional problems, stress, and chronic inflammation decrease the quantity and quality of sperm. Low sperm count, low sperm motility, and structural differences in sperm all make it harder for sperm to fertilize an oocyte [4].

Evaluation of male infertility has historically been based on semen analysis, which has been classified in accordance with World Health Organization (WHO) guidelines including sperm volume, concentration, motility, and morphology [5]. However, roughly 15% of infertile patients followed for male factor parameters have normal sperm parameters [6]. Accepted as a novel indicator of sperm quality, sperm DNA damage plays a crucial role in fertilization, implantation, and transmission of paternal genetic information to progeny [7, 8]. Recent research has focused on the possible effects of sperm DNA damage, particularly in male infertility [9]. In addition, semen samples from infertile men have been found to contain extremely reactive oxygen radicals (ROS), including hydroxyl radicals (OH), superoxide anion (O2-), and hydrogen peroxide (H2O2) [10]. It has been shown that low DNA integrity, high ROS levels, and DNA fragmentation have a big effect on male infertility [10, 11, 12].

For this reason, high-level sperm analysis methods that evaluate DNA integrity, DNA fragmentation rates, and the number of reactive oxygen species (ROS) are currently under investigation [13]. Traditional methods for selecting sperm in assisted reproduction still use motility and morphology, ignoring important factors like DNA integrity, the number of ROS, membrane maturation, and the selection of non-apoptotic sperm [10]. In the studies, it is asserted that activities such as centrifugation, pipette mixing, and washing, which are commonly utilized in conventional methods, generate ROS formation, resulting in DNA damage and an increase in the DNA fragmentation rate [14, 15]. Also, using a centrifuge to choose sperm takes time, and technicians have different ways of evaluating the results [16, 17].

Natural sperm selection in the female genital tract is influenced by a series of anatomical barriers that begin with the cervix and uterus and terminate in the uterine tube, which is where fertilization takes place. These barriers begin with the cervix and uterus and continue until they reach the uterine tube [18]. Instead of centrifugation stages that can create chemical and reactive oxygen radicals, microfluidic fluid technologies imitate the natural sperm selection pathways in the female genital tract. Thus, it is stated that fewer oxygen radicals are produced, sperm DNA fragmentation is reduced, and sperm DNA integrity is enhanced. Also, research has shown that the sperm survival rate, the sperm total motility rate, and the sperm velocity rate are better than those of other methods [14, 15].

The “Fertile Chip®,” a microfluidic liquid-based sperm selection technology, has been shown to select sperm with less DNA damage in a number of experiments now published in the scientific literature. Microinjection with sperm collected using these methods has been the subject of a small number of studies. This essay will focus on the evaluation of fertile chips to the male factor.

1.1 Assessment of male fertility

Male factor contributes to at least 50% of infertile couples and is the sole cause of infertility in 15–20% of couples [19] (Table 1). Sperm must complete normal spermatogenesis phases for conception. It includes maturation and capacitation, hyperactivation, attachment to the zona pellucida, acrosome reaction, sperm-oocyte membrane fusion, chromatin decondensation, and male–female pronucleus fusion [20]. Normal genetic structure and a normally functioning hormonal axis are essential for these processes to occur.

Pituitary Hypothalamic Causes: Isolated gonadotropin deficiency due to idiopathic causes’ Kallmann syndrome Single gene mutations (e.g., GnRH receptor, FSH or transcription factor defects involving pituitary development) Tumor of the hypothalamus and pituitary (e.g., Craniopharyngioma, macroadenoma) Infiltrative conditions (sarcoidosis, histiocytosis, transfusion siderosis, hemochromatosis) Hyperprolactinemia Medicines (GnRH analog, androgens, estrogens, glucocorticoids, opiates) Chronic disease or malnourishment Infections (e.g., meningitİs) Obesity

Primary Gonadal Conditions; Klinefelter syndrome Deletion of the Y chromosome Cryptorchidism Varicosele Inoculations (e.g., Viral orchitis, Leprosy, Tuberculosis) Medicines (e.g., alkylating agents, alcohol, antiandrogens, cimetidine) Radioactivity and environmental toxins (e.g., temperature, smoking, metals, organic solvents, insecticides) Chronic conditions (kidney failure, cirrhosis, cancer, sickle cell anemia, amyloidosis, vasculitis, celiac disease)

Sperm Transport Disorders; Epididymal obstruction or impairment Congenital bilateral absence of vas deferens (secondary to CFTR mutation) Infections causing obstruction of the vas deferens (e.g., gonorrhea, chlamydia, tuberculosis) Vasectomy Kartagener syndrome (primary ciliary dyskinesia) Young syndrome Ejaculatory dysfunction (e.g., spinal cord disease, autoimmune dysfunction)

Table 1.

Male infertility causes.

Due to a greater understanding of the male reproductive system and the significance of the male factor in infertility, the treatment of male infertility and its methodology has advanced rapidly over the past two decades. IUI can be utilized to achieve pregnancy in cases of minor male factor and IVF can be used in cases of more severe diseases [21].

1.1.1 Spermatogenesis

Spermatogenesis is the process by which sperm are produced from primordial germ cells. Approximately 75 days are required for the maturation of spermatogonia into mature sperm. Every 16 days, a new cohort of spermatogonia enters the human spermatogenesis cycle [22].

Spermatogenesis is an intricate differentiation process that begins at birth with the transformation of spermatogonial stem cells [23]. It has three phases: mitotic proliferation of spermatogonia, meiosis of spermatocytes, and haploid differentiation of spermatids [24]. Mitosis is responsible for the multiplication of differentiating spermatogonia (with 46 chromosomes). After the proliferation phase, the prophase of the first meiosis commences, during which spermatocytes remain for an extended period, homologous chromosome pairs, synapses, and homologous recombinations are formed, and homologous recombinations occur [25]. Later, the spermatocytes separate into sister chromosomes and divide into two cells, resulting in the production of secondary spermatocytes. These cells also divide very rapidly, and the resulting haploid spermatids initiate the spermiogenesis stage of differentiation. During spermiogenesis, sperm-specific structures such as the flagellum and acrosome are formed. Additionally, the nucleus condenses and the majority of histones in the DNA structure are replaced with sperm-specific protamines, causing chromatin to become dense [26]. Spermation is the process by which spermatozoa released into the tubular lumen travel to the epididymis for final maturation and storage [27]. In the epididymis, spermatozoa gain progressive movement and continue to mature for approximately 10 days [28].

The epididymis stores sperm until ejaculation. Capacitation and hyperactivation occur in the female reproductive tract [29].

FSH and LH secreted by the pituitary and stimulated by the release of hypothalamic gonadotropin-releasing hormone provide hormonal control over spermatogenesis (GnRH). In the hypothalamus, pituitary, and testis axis, a negative feedback control system exists. High serum testosterone levels inhibit the release of GnRH and LH, but physiological testosterone levels do not inhibit the release of FSH. Inhibin B, produced by Sertoli cells in response to FSH stimulation, inhibits FSH secretion at the pituitary gland [30].

1.1.2 Causes of male infertility

Male infertility can be divided into 4 major categories [21]: Hypothalamic–pituitary disorders (pretesticular disorders, secondary hypogonadism), testicular disorders (primary spermatogenesis failure and primary hypogonadism), posttesticular defects (sperm transport disorders), and idiopathic (Table 1).

1.1.3 Anamnesis

Evaluation of the male partner should begin at the same time as the evaluation of the female partner, beginning with a thorough medical history. Furthermore, the anamnesis should contain the following; infertility evaluation, genitourinary history (trauma, genital infection, difficulty sustaining an erection or ejaculating), medical record (history of high fever, chronic illness, drug use, smoking and alcohol, operation history) and family history [31].

1.1.4 Physical examination

If a gynecologist is performing the infertility evaluation, the physical examination may be delayed if the initial evaluation of the male patient does not reveal an abnormal anamnesis or a problem with the semen analysis. However, abnormal sperm analysis or an abnormal medical history is a cause for a physical examination, and the patient should be evaluated by a urologist [32].

A thorough physical examination may reveal the absence of secondary sex characteristics, suggestive of hypogonadism, or the absence of the vas deferens, a cause of obstructive azoospermia. Although physical examination should not be performed prior to sperm analysis, it is essential when there is a possibility of a problem in the clinical history or when searching for reversible causes of potentially abnormal sperm analysis parameters [31].

1.1.5 Semen analysis

Semen analysis, in the evaluation of male infertility, is the most significant parameter that provides information about the functional status of the seminiferous tubules, epididymis, and accessory sex glands [33]. A period of sexual abstinence of two to five days is required in order to obtain an optimal sample of sperm. While semen volume and density decrease when fasting periods are shortened, sperm motility and morphology do not change, and when fasting periods are prolonged, semen volume and density increase along with an increase in dead, immobile, and morphologically abnormal sperm [34]. Sperm can be collected in a sample container by masturbation or by using condoms designed for sperm collection that do not contain sperm-toxic substances. Ideal sample collection would occur in the laboratory. If the sample is collected at home, it must be transported at room temperature or body temperature and examined within one hour. The delay in the review may affect certain parameters. For instance, after two hours, there is a progressive decrease in motility as the activity of free radicals increases.

Both macroscopically and microscopically, sperm are evaluated on the basis of the following factors:

1.1.6 Macroscopic evaluation

Coagulation, liquefaction time, color, appearance, viscosity, volume, and pH are the macroscopically evaluated parameters.

1.1.7 Microscopic evaluation

Sperm aggregation: It is the result of nonmotile sperm adhering to one another or to nonsperm cells in the environment.

Agglutination of sperm: It is the coexistence of motile sperm by adhering head-to-head, tail-to-tail, or in a mixed state. It is labeled as Grades 1 through 4.

Concentration of sperm: It is the quantity of sperm per milliliter is the sperm concentration. Using a Makler counting chamber, the total number of sperm in 10 medium-sized squares is recorded as millions per milliliter. The same count is repeated four times across ten frames, and the average is then calculated. Normal sperm has a lower reference value of 15 × 10⁶ /ml [35]. While sperm concentrations below this value are associated with a poor prognosis for fertility, there is no conclusive evidence that concentrations above 15 × 10⁶ /ml improve fertility prognosis [36]. According to some sources, the probability of conception rises until the concentration reaches 40 to 50 × 10⁶ cells per milliliter, and then it remains constant [37, 38]. Severe oligozoospermia is diagnosed when the concentration of sperm is below 5 × 10⁶/ml. In the case of severe oligozoospermia, endocrinological and genetic testing should be conducted.

Total sperm number: It is the total number of sperm in the ejaculate, and the lower reference value is 39 x 10⁶. It is calculated by multiplying the sperm concentration by the volume. If no sperm cells are detected during the initial microscopic examination, the entire ejaculate is centrifuged at 3000 g for 15 minutes, and pellet drops are examined between the lamella and lamella. And if sperm cells are seen (cryptozoospermia), the total number, motility, and distinct morphological feature are recorded. A condition known as azoospermia occurs when no sperm cells can be found in the entire sperm pellet. At least two tests must demonstrate the absence of sperm.

Movement of sperm: Motility is the proportion of sperm that exhibit tail movement. After liquefaction, it must be completed within one hour.

According to WHO 2010 [39], a simple system for grading motility is recommended that distinguishes spermatozoa with progressive or nonprogressive motility from those that are immotile. The motility of each spermatozoon is graded as follows:

Progressive motility (PR): Spermatozoa moving actively, either linearly or in a large circle, regardless of speed.

Nonprogressive motility (NP): All other patterns of motility with an absence of progression, e.g. swimming in small circles, the flagellar force hardly displacing the head, or when only a flagellar beat can be observed.

Immotility (IM): No movement.

This system evaluates the proportion of sperm that fall into each category. According to the World Health Organization, a + b should exceed 40%, while an alone should surpass 32% [39]. Asthenospermia is a movement disorder characterized by a decrease in motility, forward movement, or both. In these patients, structural abnormalities of spermatozoa, long-term sexual abstinence, genital infections, anti-sperm antibodies, varicocele, partial ductal obstruction, and idiopathic factors may be to blame.

1.1.8 Sperm morphology

For the evaluation of sperm morphology, the sperm must be stained. The most common dyeing techniques are the Papanicolau method and the Diff-Quick method. WHO criteria and Kruger’s strict criteria are the most common standards for evaluating sperm morphology [40]. In order for sperm to be considered normal, its head, neck, middle section, and tail must all be normal. The proportion of sperm with normal morphology should be 14% according to Kruger’s strict criteria and > 4% according to the World Health Organization. Normal values in sperm analysis do not represent the bare minimum required for fertility. Aside from these characteristics, the male could be fertile. However, even individuals with normal sperm parameters may be infertile [41].

1.1.9 Sperm viability

Sperm viability is based on the examination of sperm cell membrane integrity, and sperm viability tests are particularly significant when the percentage of increasingly motile sperm is less than 40%. In the eosin-nigrosin or eosin-Y test, sperm with compromised membrane integrity absorb the dye and appear stained, whereas in the hypoosmotic swelling (HOS) test, sperm with intact membranes swell by absorbing the hypoosmolar fluid and their tails are curved. At least 200 sperm cells are required to determine sperm viability. The minimum acceptable reference value for sperm viability testing is 58%.

1.1.10 Nonsperm cells

In addition to sperm cells, the ejaculate contains epithelial cells of the genitourinary system, immature germinal cells, and leukocyte cells. Other cells than leukocytes are also referred to as round cells. The number of round cells and leukocytes in normal ejaculate should be 1 × 10⁶ per milliliter. If an increase in round cells is seen, a leukocyte peroxidase test or leukocyte markers should be performed to determine whether these cells are leukocytes. None of the parameters of standard sperm analysis are specific for demonstrating the fertilization capacity of sperm, and standard sperm analysis may not be adequate for distinguishing definitively between fertile and infertile sperm. Consequently, sperm function tests are required [42].

1.1.10.1 Sperm function tests

WHO accepts sperm function tests as research tests that predict the in vitro fertilization potential of sperm [42].

Computer-assisted analysis of sperm: CASA (computer-assisted sperm analysis) can be used to evaluate sperm concentration, motility, and morphology, as well as the spiral movement pattern and hyperactivation sperm acquire during capacitation [43].

Acrosome response: The acrosome is a membrane-bound structure in the sperm’s head region that contains proteolytic enzymes that are essential for penetrating the zona pellucida. One of these proteolytic enzymes is acrosine. Infertile men have a premature spontaneous acrosome reaction, which hinders zona pellucida penetration [44].

Zona pellucida (ZP): It plays a crucial role in the regulation of fertilization. The acrosome reaction is triggered by the binding of spermatozoa to the zona pellucida via the ZP3 receptor [45], which is the only physiological stimulus for the acrosome reaction. Sperm must recognize and bind to species-specific receptors in ZP for oocyte fertilization.

Both the “Hemizona assay” and the “competitive intact zona binding assay” are frequently used as zona pellucida attachment tests [43]. Due to the difficulty of locating human oocytes in both of these tests, they are not commonly used to assess male infertility.

Test for oocyte penetration in hamsters: It is used to demonstrate the success of in vivo and in vitro fertilization as a predictive test [46]. The test evaluates spermatozoal viability, acrosome reaction, ability to penetrate the oolemma, and oocyte fusion.

Test for hypo-osmolar swelling (HOST): Permeability to water is a crucial physiological characteristic of all cell membranes. Membranes permit the selective passage of liquids and molecules. The HOS test can evaluate the sperm membrane, which plays an important functional role during fertilization. There was a correlation between the number of swollen sperm in the sample of sperm and the number of sperm that successfully fertilized the hamster egg. The HOS test is predicated on the viability of spermatozoa under moderate hypoosmotic stress. Since dead spermatozoa lack intact membranes, they cannot swell. Classifying HOS-reactive cells from A to G based on the degree of swelling and tail curl. When 200 sperm are counted, the percentage is reported. Sperm with a HOS reaction of greater than 60% is considered normal. Less than 50% tail curl is considered abnormal. Acceptable is an intermediate value between 50 and 60%. HOS can be used as an additional sperm viability indicator and in the diagnosis of immotile cilia syndrome [43, 47].

Reactive oxygen radicals: Oxidative stress is one of the most important mediators in a variety of male infertility etiologies; it has many negative effects on sperm, including DNA damage. Oxidative stress occurs when levels of ROS and other free radicals are significantly elevated, when the delicate balance between oxidizing agents and antioxidants is upset, or when antioxidant levels drop significantly. Reducing oxidative stress is a possible strategy for treating male infertility. Seminal oxidative stress measurement is essential for identifying and monitoring patients who may benefit from treatment [48].

Mitochondrial activity tests: Spermatozoa obtain the energy necessary for flagellar movement from adenosine triphosphate (ATP) produced by mitochondria in the middle portion of spermatozoa. Spermatozoa require a sufficient amount of mitochondrial apparatus in the female genital tract in order to produce the necessary ATP during their journey to the oocyte. For the demonstration of the mitochondrial oxidoreductase enzyme, nitro blue tetrazolium and similar indicators are used. With these indicators, the middle portion of motile sperms with abundant mitochondria is prominently stained, whereas the middle portion of immobile sperms with low mitochondrial activity is either not stained at all or is stained less. Their staining revealed a statistically significant correlation between mitochondrial activities and sperm motility [43].

DNA damage tests: These are crucial for ensuring normal embryo development. The effect of disulfide cross-links between protamines, which provide chromatin condensation in the nucleus, partially preserves the integrity of sperm DNA. Sperm DNA damage can be caused by internal factors like protamine deficiency and mutations, or by external factors like heat, radiation, and gonadotoxins. The term “DNA fragmentation” refers to irreparable denatured or damaged sperm DNA. Various clinical tests for measuring sperm DNA fragmentation rates have been developed [19]. Over the years, an increasing number of sperm DNA integrity tests have been developed. The mechanism for evaluating DNA integrity in these tests varies. While some tests directly measure breaks in the DNA helix, others reveal abnormalities in sperm chromatin structure [48, 49]. DNA damage in male germ cells appears to be linked to poor sperm quality, impaired preimplantation development, an increased risk of miscarriage, and infertility [50].

1. Nuclear chromatin decondensation test: Although sperm contains half as much DNA as a typical eukaryotic cell, its volume is only one-thirtieth as large. Due to the reduction in volume, DNA packaging is a very complicated process. The chromatin of spermatozoa is highly condensed before fertilization. Fertilization requires appropriate nuclear chromatin decondensation and subsequent formation of pronuclei. Spermatozoa’s highly condensed chromatin is due to the S-S bonds between histones. EDTA (ethylenediaminetetraacetic acid) and glutathione can induce dissociation between bonds in vitro. This method of inducing decondensation is indicative of the fertility of spermatozoa. More than 70% nuclear decondensation in sperm is considered normal [51].

2. DNA fragmentation index: DNA damage can be evaluated directly by DNA fragmentation using the TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate) assay, SCSA (sperm chromatin structure assay), sperm chromatin distribution, or comet assay. The percentage of sperm with DNA fragmentation was negatively correlated with normal sperm morphology and motility. DNA fragmentation may be caused by advanced male age, genetic causes, environmental toxins, endocrine disorders, alcohol, tobacco use, and diet.

1.2 Assisted reproductive technology (ART)

The majority of assisted reproductive techniques facilitate conception in a laboratory to assist infertile couples in having children. Intrauterine insemination (IUI), in vitro fertilization (IVF), and intracytoplasmic sperm injection (ICSI) are the most common assisted reproductive technologies (ARTs) [52]. In preparation for ART, spermatozoa must be collected from the male. After collecting sperm and washing them using swim-up or density gradient centrifugation techniques, the motile sperm are selected. During the ovulation phase of IUI, spermatozoa are introduced into the uterus. Following stimulation of follicular development in the ovaries, primary oocytes are collected for IVF and ICSI. Multiple oocytes are extracted in order to produce multiple embryos for implantation. The use of sperm and oocyte to complete IVF or ICSI [39].

1.2.1 Intracytoplasmic injection of sperm (ICSI)

ICSI was developed primarily to facilitate fertilization in cases where sperm motility or morphology prevented spermatozoa from crossing the zona pellucida. This method involves injecting a spermatozoon directly into the cytoplasm of the oocyte to facilitate fertilization [53].

ICSI, on the other hand, is problematic because spermatozoa no longer face the obstacles they do during natural conception and are not subject to natural selection. For a successful pregnancy to occur, a viable spermatozoa with good DNA must be injected into the oocyte since natural selection is inhibited by this method [54]. Since there is no natural selection of sperm, selecting the appropriate sperm is crucial.

1.2.2 The importance of sperm selection in ICSI treatment

Despite decades of widespread use of ART to treat infertility, live birth rates remain relatively low [55]. According to the Centers for Disease Control and Prevention (CDC), it is unknown why so many insemination attempts fail to result in fertilization, while only a third of many ART cycles result in a live birth [56]. Infertile men have abnormal sperm parameters, including low sperm concentration, poor motility, abnormal morphology, and elevated sperm DNA damage levels. Low ROS concentrations are necessary for essential sperm functions such as capacitation, hyperactivation, and acrosome reaction, and excessive ROS production is typically regulated by antioxidants [57]. High levels of ROS and low levels of antioxidants can cause oxidative stress, which reduces sperm motility, DNA integrity, and viability. Reduced DNA integrity leads to decreased IVF pregnancy rates, an increase in preimplantation developmental abnormalities, and an increase in early pregnancy loss [57].

Very few of the millions of sperm that are poured into the vagina reach the oocyte. Naturally occurring in the female genital tract, this is a very precise and flawless elimination system. As sperm reach the vagina, vaginal mechanical stimulations support the sperm’s swimming movement, directing them toward the uterus and tuba. In the storage area, sperm undergo a maturation process known as capacitation. The mature sperm move toward the oocyte via chemotaxis and thermotaxis following capacitation. Sperm penetrate cumulus cells as a result of chemotaxis, bind to sperm receptors in the oocyte, and initiates the acrosome reaction. Consequently, fertilization occurs [58].

Today, sperm selection techniques for ART bypass the barriers of natural selection and select sperm based primarily on motility and morphology, ignoring other important factors such as DNA integrity, ROS production, membrane maturation, and non-apoptotic sperm selection [18]. In addition, traditional sperm selection techniques such as density gradient centrifugation (DGS), conventional swimming (CSW), and direct swimming (DSW) generate high levels of ROS through the use of multiple centrifugation steps, resulting in DNA damage due to oxidative stress [59]. According to clinical data, a DNA fragmentation index above 30% reduces the likelihood of natural and artificial conception [60].

Additionally, while fertilized oocytes have DNA repair mechanisms, spermatozoa do not, so they cannot repair DNA breaks after spermatogenesis [60]. Therefore, in order to select sperm with normal DNA and fewer ROS and to increase ART success rates, it is necessary to develop new sperm selection techniques in addition to enhancing existing ones. To ensure that healthy sperm are selected, new sperm selection methods must closely mimic the natural selectivity of the female genital tract.

1.2.3 Conventional sperm selection techniques

Traditional methods for sperm selection involve multiple washing and centrifugation steps. Density gradient centrifugation (DGS), conventional swim-up (CSW), and direct swim-up are the most frequently employed conventional sperm selection techniques (DSW).

Density gradient centrifugation (DGS), conventional swim-up (CSW), and direct swim-up are the most frequently employed conventional sperm selection techniques (DSW).

• Density gradient centrifugation (DGS) technique: It enables the selection of high-quality sperm and their differentiation from other cell types and cell debris. As it is simpler to standardize than the swim-up technique, the results are more consistent. This method is used to retrieve sperm for IVF and ICSI. This technique involves the centrifugation of seminal plasma based on density gradients. Using density gradients containing colloidal silica-coated silane, this method separates cells based on their density. Furthermore, motile sperm swim through the gradient material and form a soft pellet at the tube’s bottom. The most common simple two-stage discontinuous density gradient preparation method consists of an upper layer with a density of 40% (v/v) and a lower layer with a density of 80% (v/v). Using density gradient centrifugation to prepare sperm typically results in highly motile sperm free of cell debris, contaminating leukocytes, nongerm cells, and degenerative germ cells [39].

• Direct swim-up (DSW) technique: Sperm can be chosen based on their ability to swim from the seminal plasma into the culture medium. This technique is known as the “swim-up” method. Prior to employing this method, sperm should ideally not be diluted or centrifuged. Otherwise, sperm membranes may be susceptible to peroxidative damage [61]. Therefore, direct flotation is the preferred technique for separating motile sperm. Direct flotation can be accomplished by either spreading the culture medium on top of the liquefied sperm or by spreading the liquefied sperm as a layer beneath the culture medium. Next, motile sperm enter the culture medium. Although this method yields fewer sperm than washing, it is useful when the proportion of motile sperm in the sperm is low (e.g., for IVF and ICSI) because it selects sperm based on their motility [39].

• Conventional swim-up technique (CSW): Before incubation, sperm are precipitated in the conventional swim-up technique by centrifugation. Then, using a pipette, the 1 ml portion floating on the top is removed and utilized. It is a technique that relies solely on sperm motility. Asthenozoospermia and oligozoospermia may render it inappropriate. In cases of severe male infertility, its use is therefore restricted.

Although conventional methods are effective at selecting motile and morphologically normal sperm, they are insufficient for selecting sperm DNA integrity, membrane maturation, detailed structural characteristics, and non-apoptotic sperm [62].

1.2.4 Advanced sperm selection methodologies

Zeta Potential: Approximately between −16 mV and − 20 mV, the zeta potential of sperm is the electrical potential between the sperm membrane and its surroundings. Using a latex glove, the sample of washed sperm is pipetted into the positively charged centrifuge tube and gently mixed in the tube two to three times. After one minute of centrifugation, Sperm and other cells that do not adhere to the edge of the tube are removed (Figure 1). Since no electrophoresis equipment is required, the zeta method is inexpensive and simple to employ. Additionally, the Zeta treatment has been successfully applied to freeze-thawed sperm samples [63]. However, its effectiveness in oligozoospermic samples with a low sperm count is limited. When electrophoretic methods are compared to the DGS method, it has been observed that the sperms obtained have a high level of maturity, morphology, and DNA integrity, but their motility is low [63, 64].

Figure 1.

Separation of sperm by zeta potential.

MACS: Magnetic activated cell sorting system early apoptosis is characterized by the externalization of phosphatidylserine (PS), which is located on the outer surface of the sperm membrane. Utilizing a MACS, the selection of nonapoptotic sperm is achieved in this method [65]. Annexin V binds to paramagnetic microbeads that mark and separate apoptotic sperm in the event of PS externalization. A heterogeneous concentration of sperm cells is initially incubated with microbeads conjugated with Annexin V; however, only apoptotic sperm with externalized PS bind to these beads. The mixture of beads and sperm is passed through a MACS column equipped with a magnet. This magnet retains microbead-labeled cells in the interior of the colon and ensures their gradual removal by a steady flow of unmarked cells [66]. Due to the inability of MACS to remove leukocytes and germ cells, this technique is utilized in conjunction with DGS [67] (Figure 2). Recent ICSI studies comparing sperm samples prepared with or without MACS revealed no statistically significant differences in implantation, miscarriage, or live birth rates [68]. Before concluding that this technique is effective in ICSI procedures, it should be evaluated in studies with larger sample sizes, using a larger number of samples.

Figure 2.

Separation of sperm by MACS. Loading the tubes into the device (A), putting Annexin V-labeled apoptotic sperm and non-apoptotic sperm into the tubes (B), magnetic capture of Annexin V-bound apoptotic sperm and advancement of non-apoptotic sperm into the collection tube (C).

Hyaluronic acid adherence: Hyaluronic acid (HA) is the primary constituent of the cumulus oophorus’ extracellular matrix. Binding sites for hyaluronic acid in the sperm plasma membrane indicate sperm maturity. There are two ways to select HA-bound sperm: physiological intracytoplasmic sperm injection (PICSI) and the sperm-slow procedure. Both methods require sperm washing or centrifugation. In order to select sperm, a product called “PICSI dish” with four HA-fixed compartments has been developed. A drop of the washed sperm is placed on the edge of the HA spot, and after 15 minutes, the HA-bound sperm are collected with an injection pipette and used for ICSI [69] (Figure 3). Additionally, HA binding is commonly used to select mature sperm with a low frequency of chromosomal abnormalities. This increases the likelihood of genetic complications following ICSI. In a study of semen samples from men undergoing fertility treatment, it was discovered that autosomal disomy, diploidy, and sex chromosome disomy were significantly lower in HA-linked sperm than in non-binding sperm [69].

Figure 3.

Sperm appearance in the PICSI petri dish.

Electrophoresis-based sperm selection: Electrophoresis (Microflow CS-10) is a technique that selects sperms based on their surface charge. Normally, mature sperm are negatively charged due to the presence of CD52 and glycosylated phosphatidylinositol on their surface. The electrophoresis device is a cassette in which a semen sample is placed, a voltage is applied, and morphologically normal, negatively charged sperm move across a 5 m polycarbonate membrane toward the positive electrode, leaving immature sperm and leukocytes behind [70]. DNA integrity, sperm morphology, and motility were not significantly different between DGS and electrophoresis. In addition, because there is no centrifugation step in sperm selection by electrophoresis, there is less oxidative DNA damage due to the decreased exposure to ROS [70] (Figure 4).

Figure 4.

Separation of sperm by electrophoretic method-MicroflowCS-10.

Morphological evaluation of motile sperm organelles (motile sperm organellar morphology examination; MSOME): Examining the morphology of sperm under high magnification microscopes allows for the morphological evaluation of motile sperm organelles-based sperm selection. MSOME is applied at up to 6300x magnification, whereas standard ICSI is performed at 600x magnification. In this technique, which was developed by Bartoov et al. the structural characteristics of sperm are investigated in depth. To determine the healthiest sperm, the Acrosomal region, Post-Acrosomal region, Neck, Mitochondria, Flagella, Tail, Vacuole areas, and the ratio of these vacuole areas to the head are calculated [71]. MSOME has been used in conjunction with standard ICSI procedures and is named after intracytoplasmic morphology-selected sperm injection (IMSI) (Figure 5). It plays a crucial role in sperm selection for men with severe infertility.

Figure 5.

Sperm selection for IMSI.

Birefringence: Birefringence of sperm is evaluated using an inverted microscope equipped with polarized lenses. Using double refraction, sperm with reactive acrosomes can be selected during ICSI without compromising motility or viability [72]. Sperm with birefringence can be selected for microinjection, and the quality of these sperms appears to be high. A significant positive correlation exists between the proportion of birefringent sperm and other sperm parameters, including concentration, motility, and viability [72]. Similar to MSOME and IMSI, polarized microscopy for sperm selection requires additional equipment, time, and technical expertise. Comparing the microinjection method performed by evaluating sperm birefringence and routine ICSI, a high pregnancy rate and decreased miscarriage rate were observed with this new method in patients with heavy male factor [72].

Selection of sperm using a microfluidic liquid model: “Microfluidic channel system (spermchip)” is one of the methods developed for sperm selection that can prevent sperm losses and DNA damage caused by conventional sperm preparation methods. In developing this technique, the path followed by sperm during natural conception served as a model. This system includes a microchip with microchannels that mimics the intrauterine, cervical, and vaginal canal microenvironments of sperm. Microfluidic channels are formed in the microchip by a 1.5 mm thick combination of Polymethylmethacrylate (PMMA) and a 50-micron thick double-sided adhesive (DSA) film. The integration of a microchip-coupled device (CCD) into the chip enables the automatic recording of sperm movement within the microfluidic channel. Incorporating the integrated system into the microfluidic channel. The microfluidic channel medium was pre-filled with serum-supplemented human tubal fluid (HTF) medium. The sperm sample is pipetted into the column at the top channel entrance using a pipette. Sperm are anticipated to swim from duct systems of a particular length.

ICSI involves the collection and use of floating sperm. Moreover, since the microchip can be placed on the integrated device (CCD), the sperm’s shadow movement can be monitored and recorded [73]. (Figure 6). Microfluidic fluid technologies mimic the natural sperm selection pathways that take place in the female genital organs, rather than centrifugation steps that can generate ROS. Thus, it is stated that fewer oxygen radicals are formed, DNA fragmentation of sperms is lower and their DNA integrity is higher. In addition, studies have shown that sperm viability rate, sperm total motility rate, sperm velocity rates are higher than other methods [14, 15].

Figure 6.

Microfluidic channel system. Filtered motile sperm; semen sample; (a) the photo of the MSS showing inlet, filter and two PMMA chambers. The MSS was filled with color dye to enhance contrast; (b) the illustration demonstrates the MSS design and working principle. The MSS consists of one inlet for the injection of raw unprocessed semen sample and two PMMA chambers separated by nucleoporin track-etched membrane filter. The most healthy and motile sperm swim through the filter leaving unhealthy dead sperm in the bottom chamber; (c) SEM images of polycarbonate nuclepore track-etched membrane filters of different microspore diameters, i) 3 μm ii) 5 μm and iii) 8 μm. These images show the comparative size of various filter pores and sperm. The scale bar is 10 μm.

2. Discussion

Evaluation of male infertility requires a thorough examination of sperm. Significant determinants of the success of ART include sperm motility, morphology, viability, DNA integrity, apoptosis, and maturation. Recent research has revealed that the DNA integrity of sperm is essential for normal fertilization and embryo development. As a result, improved sperm selection techniques are used to identify higher-quality and healthier sperm for ICSI treatment. Although it has been established that the vast majority of these new sperm selection approaches outlined before can select for sperms with a greater DNA integrity and a lower DNA fragmentation rate, this is not the case for all of these techniques. Patients with male factor infertility who underwent IVF were studied in a prospective randomized controlled trial that compared the effects of microfluidic sperm selection technologies to the conventional swim-up strategy. Fertilization rates and embryo quality, which were among the key findings of this study, were comparable across the two groups, as evidenced by the results of this randomized controlled experiment. The study group had a greater rate of live births, implantation, and clinical pregnancy [73]. The fact that this study found statistically significant differences in the rates of implantation, pregnancy, clinical pregnancy, and live birth makes it an impressive piece of research. Despite having comparable numbers of grade 1 and 2 embryos, the control group had more grade 3 embryos. This may suggest that the Fertile Chip was used to select sperm of a higher quality, or that other parameters influencing embryo quality are not reflected in sperm morphology.

The microfluidic sperm sorting chip is simple to use, inexpensive, chemical-free, mechanical-free and perturbation-free, and it removes the centrifugation stage. At an ideal time point, the most motile and functional spermatozoa with the correct structure, high DNA integrity, and a low ROS level can be selectively passed via the microchannels of a microfluidic sperm sorting device, leaving behind the less motile or immotile spermatozoa [14].

The increase or decrease in DNA fragmentation levels observed in gradient technique applications, as well as the heterogeneity of the results reported in previous studies, may be attributable to initial cellular DNA fragmentation rates or centrifugation. In a 2018 study, Quinn et al. compared traditional sorting procedures to microfluidic chip approaches. DNA fragmentation rates utilizing the microchip method were much lower than those using the gradient method, according to the study’s findings [74]. The primary purpose of IVF treatment, however, was not evaluated in this study. Yang et al. discovered a statistically significant difference in embryo implantation rate between infertile individuals with high sperm DNA fragmentation index (DFI ≥15%) values and those with low (DFI < 15%) DFI-ICSI values (p < 0.01). There were no significant differences in fertilization rates, embryo quality, or blastocyst development [75]. Despite the lack of a statistically significant difference in embryo quality, the difference in implantation rate suggests that morphological parameters and DFI alone are insufficient to evaluate embryo quality.

When sperm separation was performed using the microfluidic platform, a small cohort of couples undergoing ICSI achieved pregnancy rates of 58.8% and implantation rates of 34.5%, according to a study by Parella et al. In the same study, it was believed that this was because the use of microfluidic sperm selection increased the likelihood of producing euploid embryos [76].

Using density gradient selection and microfluidic sperm sorting, Parella et al. evaluated a novel method for choosing spermatozoa with intact chromatin. This work demonstrates that microfluidic selection produces spermatozoa with high genomic integrity and increases the possibility of producing euploid embryos [77].

Green et al. explored if sperm DNA fragmentation (SDF) in the ICSI sample affects the results of euploid blastocyst transfer. According to the findings of this study, SDF levels on the day of ICSI were not associated with embryological or clinical outcomes following euploid blastocyst transfer.

Increased SDF levels are associated with lower sperm concentration and number of motile sperm [78]. Given that the transferred embryos in this study were euploid embryos with good DNA integrity, it was not anticipated that we would assess the effect of DFI. As preimplantation genetic diagnosis (PGD) is a more invasive operation, and for individuals who cannot undergo PGD for budgetary reasons, the microfluidic technology can be used to pick a more capable embryo. With a sperm DFI > 20%, the clinical pregnancy rate of IVF-ET was significantly reduced, while with a sperm DFI > 30%, the rate of available embryos decreased significantly and the biochemical pregnancy rate increased dramatically, according to a study report published in the current scientific literature. There was no correlation between sperm DFI and fertilization, embryo cleavage, or high-quality embryo rates in IVF-ET. A high DFI reduced the pregnancy rate without impairing embryo quality [79]. By simulating the natural pathways that choose healthy spermatozoa traveling via the cervix, uterine cavity, and fallopian tubes, microfluidic selection may be useful in selecting higher-quality spermatozoa.

In the current literature, one study examined the effects of using microfluidic chips vs. gradient-density centrifugation for sperm selection in ICSI cycles in male infertility patients. According to the findings of this study, there were no statistically significant differences between the groups in terms of CPR and continued PR, although they were significantly higher in the group using microfluidic sperm sorting chips for male infertility [80]. In couples with a total motile sperm count between 1 and 5 million, the rise in pregnancy rate was more substantial (p < 0.01). Nonetheless, this was a retrospective study in which the spermatozoa of the study group had very poor morphology and the groups were not homogenous.

Guler et al. have conducted a prospective, randomized, controlled study evaluating the impact of density gradient centrifugation and microfluidic chip sperm preparation techniques on embryo development in astheno-teratozoospermia patient populations. Although the density gradient group had a higher sperm concentration, the microfluidic chip group had much greater motility (progressive and total). On the third day, there were no significant differences in fertilization rates or proportions of grade 1 and grade 2 embryos, as determined by the research. In addition, whereas the proportions of poor, fair, and good blastocysts on day 5 did not differ significantly, the microfluidic chip group had a much higher proportion of exceptional blastocysts (indicating high-quality embryos). The microfluidic chip sperm preparation produced sperm with greater motility and higher quality blastocysts on day 5, in patients with asthenoteratozoospermia [81].

3. Conclusion

The different procedures presented in this chapter for human sperm selection have advantages and disadvantages, and as described, several of these strategies have produced inconsistent results, leaving their therapeutic usefulness uncertain. Among these processes, standard sperm separation techniques (swim-up and DGC) are the most commonly utilized in ART laboratories, despite the uncertainty surrounding their deleterious effects on sperm cells. Due to the “excellent” characteristics of selected sperm, however, microscopy-based selection approaches are becoming increasingly popular. However, microscopy-based approaches and some of the other mentioned technologies are too costly or technically sophisticated to be utilized in ordinary ART settings.

The optimal sperm sorting process for ART should efficiently separate healthy, motile, and morphologically normal sperm that are capable of fertilizing oocytes. In contrast to conventional sperm-separating methods, which require many centrifugation steps to retrieve sperm cells, the optimal sperm sorting strategy should not use centrifugation. In fact, it has been demonstrated that sample centrifugation induces sperm cell ROS generation and DNA fragmentation. Importantly, the process must be non-invasive, as the same sperm extracted based on one or more functional features must be utilized for fertilization. In addition, the embryologist must select the appropriate sperm selection procedure based on the infertile status of the patient, such as oligospermia or obstructive azoospermia, as well as sample quality. Therefore, it is quite difficult to choose a single strategy from those described in this review. As a result, several laboratories combine multiple approaches to improve the quality and quantity of picked sperm. Insufficient randomized controlled studies and meta-analyses exist to aid the embryologist in making a decision. In this regard, lab-on-chip systems offer a number of practical benefits, including the ability to sort sperm through improved automated methods and reduce sperm losses caused by complex protocols and multiple transfers. Studies demonstrated that chips for sperm sorting are simple to use, economical, do not require several manual stages, and are not dependent on embryologist skill, hence eliminating human error and permitting standardization of sperm separation for assisted reproductive technology (ART) treatments.

Considering the above-mentioned promising results, such labs-on chips are expected to soon become more commonly used in infertility treatment centers around the world.