Semen Analysis, Techniques and Evaluation

#### **Chapter 2**

### Semen Analysis and Infertility

*Suchada Mongkolchaipak*

#### **Abstract**

Male factor infertility contribute approximately at 50% for the cause of infertility. The steady declination of semen quality in men for all over the world might be from various factors such as life style changes, environmental toxicity, dietary contribution and social problems. Assisted reproduction is the main treatment of choice for male infertility; However, in severe male factor infertility, the treatment outcomes could end up with recurrent implantation failure or recurrent pregnancy loss. Basic semen analysis still has limitation to explain the cause of failure for the part of male factors. The purposes of developing new sperm evaluation methods are to improve the diagnostic tools for identifying the sperm defects, appraise of fertility potential and provide suitable treatment for an infertile couple, explain the cause of treatment failure from male factor part and measure the efficacy of male contraception.

**Keywords:** male factor infertility, sperm DNA fragmentation, sperm oxidative stress, semen analysis, total antioxidant capacity, sperm motility, sperm morphology, sperm concentration

#### **1. Introduction**

According to the World Health Organization (WHO), infertility is a reproductive system disease that is defined as "a failure to conceive naturally after regular sexual intercourse without the use of contraception for at least a year" [1]. It is estimated that 8–12% of people who are of reproductive age encounter infertility problems [2]. The male factor is responsible for 50% of infertility cases [3].

Furthermore, the incidence of male factor infertility in men in the active reproductive age bracket has been reported to be as high as 15% [4]. The causes of male factor are multifactorial. The pathophysiology of the causes is not fully understood, and most of them are idiopathic [5]. Initial investigation of the male partner consists of a history, physical examination, and semen analysis; however, some may require specific hormonal investigations to determine the causes of male infertility [4].

Semen analysis is a basic method to investigate the cause of infertility in the male. Six versions of a semen assessment methods guideline have been created by WHO. The first manual was published in 1980 based on clinical experience and research. The following versions were published in 1987, 1992, 1999, 2010, and 2021. The reasons for revising the manual included improving the semen analysis methods and updating the semen parameters to be more compatible with normal male fertility [6–9].

Nevertheless, at least 15% of infertile men are found to have a normal semen analysis according to WHO criteria [10]. However, further abnormalities were detected by a sperm DNA integrity test [11–13]. In the latest edition of WHO's laboratory manual for human semen evaluation, revisions have been made to improve the accuracy of the test and eliminate the unnecessary steps of the evaluation [14].

#### **2. Semen sample assessment**

Semen analysis is the method of evaluating the ejaculate composed of sperm, which originates from the testes and seminal fluid secreted from the accessory glands. Components in seminal fluid facilitate the sperm's access to the female genital tract and its ability to fertilize the mature oocyte *in vivo* [15]. In the sixth edition of WHO's laboratory manual for human semen examination, the procedure is divided into three parts. The first part is a basic semen examination. The second is an extended analysis, which is specialized for specific clinical applications. The last part is an advanced procedure, which is not used in routine practice. It must be done in a special laboratory, and it is mainly used in research studies [16]. The basic assessment includes the measurement of ejaculate volume, macroscopic evaluation, and microscopic examination.

In the sixth WHO manual, revisions to the basic assessment are based on evidence-based practices that improve the process of assessment, reduce the workload in the laboratory, and promote inter-laboratory quality assurance. The WHO manual emphasizes the importance of precisely measuring the ejaculate volume, which reflects the true total sperm count and examining the extent of sperm motility, which is clinically related to the male fertility potential.

#### **3. Basic examination of semen sample parameters**

General semen quality has shown a steady decline across the world [17] due to various factors, such as lifestyle changes, environmental toxicity, dietary contribution, and social problems [18–20]. Today, there are multiple tools to evaluate the semen quality and find suitable treatments for each infertile couple. Each semen parameter reflects the individual cause that needs to be clarified.

#### **3.1 Gross appearance**

Normal ejaculate has a homogeneous grayish color. In the case of pale color or colorlessness, the patient should be asked if there is orgasm during seminal collection because the semen component might only be from the Cowper's glands, not the prostate gland, seminal vesicle, or seminiferous tubule. The former occurs from sexual arousal, while the latter occurs from an orgasm. The color could be a deep yellow in patients with jaundice or those taking vitamin supplements. Normally, the ejaculate is odorless and can be liquefied within 30 min at room temperature. A strong smell is not normal and should be noted in the report.

#### **3.2 Volume**

The ejaculate volume is clinically significant for the diagnosis of male infertility, as it reflects the total sperm count in the ejaculate. The sixth WHO manual focuses on

#### *Semen Analysis and Infertility DOI: http://dx.doi.org/10.5772/intechopen.107625*

the accuracy of the volume measurement by instructing to weigh the ejaculate (preweighing the container and subtracting it from the total weight of the final specimen) and calculate back into the volume. The formula for calculating semen density is 1 g per 1 ml. Total sperm count is related to the sperm produced directly from the seminiferous tubules. This count is used as an indicator of spontaneous conception and treatment success in intrauterine insemination [21].

#### **3.3 Microscopic examination**

#### *3.3.1 Sperm concentration*

The clinical value of sperm concentration is less than the total sperm count per ejaculate because the sperm concentration depends on the amount of accessory gland secretion activity. Sperm concentration does not indicate the chance of success in intracytoplasmic sperm injection (ICSI) cycles [22].

#### *3.3.2 Sperm motility*

In the sixth WHO manual, the assessment of sperm motility has reverted to the four categorizations presented in the fourth WHO manual: rapidly progressive (≥25 μ m/s), slowly progressive (5 to <25 μ m/s), nonprogressive (<5 μ m/s) and immotile (no active tail movement) (they are classified as grade a, b, c, and d, respectively) movement. However, in the fifth edition, sperm motility was classified into just three categories: progressive motility, nonprogressive motility, and immotile.

The total number of progressively motile spermatozoa indicates the chance of success in intrauterine insemination [23], and the presence of rapidly progressive motile sperm is clinically significant [24]. Asthenozoospermia is a medical term defined as "lower sperm motility than the reference values." It could result from several factors.

#### *3.3.2.1 Varicocele*

Varicocele is a chronic disease involving the pampiniform plexus of veins. It creates the tortious vessels in the spermatic cord [25]. The mechanisms involved in sperm function in the case of varicocele have not been clearly explained. However, some mechanisms might be related to sperm motility. The most likely pathophysiologic cause of sperm impairment is increased scrotal temperature caused by tortious veins, which increase oxidative stress, and reflux of toxic substances from the kidneys and adrenal glands into the testes [26]. There is evidence that varicoceles impair sperm motility [27–29]. While a varicocelectomy can improve sperm motility, other sperm parameters are still controversial [30–33]. Adjuvant therapy using antioxidant supplements to improve sperm quality [34, 35] has had conflicting outcomes [36].

#### *3.3.2.2 Sexual abstinence*

The duration of abstinence impacts sperm quality, including sperm motility [37]. Consequently, the male partner should be advised to be abstinent for 2–7 days to maintain sperm analysis accuracy among patients and between laboratories [16]. The sperm kinematics was improved when the duration of abstinence was 2 hr. compared to 4–7 days, both in normal semen [38] and oligozoospermic semen [39].

However, contradictory outcomes were still reported, as some studies revealed that the duration of abstinence at 4–5 days had higher sperm motility than 2–3 days and 6–7 days of abstinence [40].

#### *3.3.2.3 Lifestyle factors*

A recent systematic review of the literature shows that smoking is a strong factor impacting sperm concentration and motility [41]. Even a moderate amount of smoking can impair progressive motility. The pathophysiology of tobacco that results in diminished sperm motility could be oxidative stress that leads to the axonemal and mitochondrial damage on the midpiece of the spermatozoa [42, 43]. Both smoking and alcohol consumption had a detrimental effect on sperm motility and other sperm parameters [44]. When considering alcohol consumption alone, it is shown to have some association with decreased sperm quality when the amount of consumption is significant and chronic [45]. In men with obesity, sperm quality is diminished in concentration and morphology but not sperm motility [46].

#### *3.3.2.4 Genetic causes of male infertility*

Some genetic defects, such as Kartagener's syndrome and primary ciliary dyskinesia, directly cause abnormal sperm motility due to their effects on the flagellar structure and function [46–48].

#### *3.3.2.5 Mitochondrial DNA mutation*

Mitochondria are the bioenergetic source for sperm activity. They are required for natural conception and *in vitro* fertilization. The mitochondrial DNA mutation could be one cause of male infertility [49–51]. Mitochondrial DNA is vulnerable to damage from reactive oxygen species due to the lack of histone protein. It is physically associated with the inner mitochondrial membrane, where free oxygen radicals are generated [50]. Recently, an association was discovered between the single nucleotide variants of the mitochondria cytochrome B gene (MT-CYB) and male infertility [52].

#### *3.3.2.6 Anti-sperm antibody*

The presence of an anti-sperm antibody in the ejaculate is correlated with semen quality—sperm count, motility, and morphology in terms of oligoasthenoteratozoospermia [53, 54]. However, the screening of an anti-sperm antibody test before ICSI is not meaningful because the process of ICSI already bypasses the natural ability of sperm to fertilize the oocyte [55].

#### *3.3.2.7 Medication's effects on sperm motility*

Several kinds of medications have deleterious effects on spermatogenesis. For example, chemotherapeutic drugs have a strong negative effect on sperm production [56–58]. However, the effect of chemotherapeutic drugs on sperm motility is still controversial. While psychotherapeutic drugs, such as imipramine hydrochloride, chlorpromazine, trifluoperazine are commonly used medications, there is strong evidence that they negatively affect sperm motility [59]. Additionally, acetaminophen, which is used as an antipyretic drug, and non-steroid anti-inflammatory drugs (NSAIDs) affect sperm motility [60, 61]. Moreover, regular consumption of marijuana also results in decreased sperm motility [62].

#### *3.3.2.8 Heat exposure*

Normal spermatogenesis requires environmental temperatures to be 32 to 35°C, which is lower than human core temperature, around 2–5°C. Research in animal models has shown that that heat stress could impact sperm motility by downregulating mitochondria activity and decreasing ATP activity [63]. Furthermore, increasing whole body temperature could induce damage to the epididymal spermatozoa's membrane, resulting in apoptosis [64]. These findings support the conclusion that heat exposure can damage spermatozoa productivity and function.

#### *3.3.2.9 Environmental factors*

Due to the rapidly growing industrial and agricultural countries around the world, the environment is polluted with pesticides, herbicides, petrochemical agents, and volatile organic compounds. These are all endocrine-disrupting agents that can interfere with normal spermatogenesis and male endocrine function. Several published data support that pesticides [65], dioxins [66], phthalates [65], perfluorinated compounds [67], polychlorinated biphenyls [65], heavy metals [68], dichloro-diphenyl trichloroethane [69], and plasticizers [70] impact sperm motility.

#### *3.3.3 Sperm morphology*

Sperm morphology is an important indicator of male fertility. Teratozoospermia is the nomenclature in the fifth WHO edition that means "lower sperm morphology than the reference value."

In the sixth edition, there is no nomenclature such as teratozoospermia to clarify the semen quality (the sperm morphology is less than the reference value). However, the editors used the lower fifth percentile value of the sperm from men with a female counterpart who has had a spontaneous pregnancy within a year without contraception with a 95% confidence interval. In clinical practice, the clinician might need to use the reference value to discriminate between fertile and infertile men, as demonstrated in the fifth WHO edition that the normal morphology is less than 4% according to the strict Kruger's criteria [9]. The sperm morphology alone cannot be used to predict the success of intrauterine insemination [71]. Therefore, in teratozoospermia without other sperm abnormalities, the couple should not be excluded from the process of intrauterine insemination. In contrast, in a retrospective study in 22,000 assisted reproductive technologies cycles, there was a predictive value of sperm morphology with fertilization rate, clinical pregnancy rate, and live birth rate [72].

#### *3.3.4 Sperm vitality*

The sperm vitality test is not a routine step in the sperm assessment process. Vitality tests should be done in semen samples that have very low motile sperm. The purpose of sperm vitality is to distinguish the immotile living sperm from the immotile dead sperm (necrozoospermia). A high percentage of dead sperm in the ejaculate indicates pathology in the epididymis (testicular cause) [73, 74] or sperm damage from infection (extra-testicular cause) [75]. The vitality can be assessed by an eosin-nigrosin (E-O) stain by evaluating sperm membrane integrity and permeability. The hypoosmotic swelling test is used to directly test the viability of the sperm without staining and evaluate the sperm membrane permeability [76].

#### **4. Potential extended and advanced examination of semen sample parameters**

#### **4.1 Sperm DNA fragmentation**

Conventional or basic semen analysis is used to identify male factor infertility. However, at least 15% of the infertile male partners have normal semen analysis based on conventional semen analysis [77, 78] However, basic semen analysis cannot detect some additional issues related to the fertilization rate, embryo development rate, and success rate in *in vitro* fertilization. Further investigation should be done on semen. Sperm DNA integrity is necessary for reproducing healthy offspring from one generation to the next generation. A DNA integrity test is a biomarker of intact chromatin and one of the independent tests available in male infertility besides basic semen analysis [79].

Reactive oxygen species (ROS) are free radicals of oxygen-producing hydroxyl radicals, superoxide anion, and hydrogen peroxide. During natural conception, low levels of reactive oxygen species are needed to maintain sperm capacitation, hyperactivation, acrosome reaction, and fertilization. DNA fragmentation occurs when there are more reactive oxygen species in the spermatozoa environment than the natural seminal antioxidant [80]. Some other external factors that impact the DNA fragmentation rate are as follows: obesity, psychogenic stress, smoking, alcoholic consumption, medication, and advanced paternal age [81].

DNA fragmentation is an important factor in detecting further male factor beyond basic semen analysis, which plays a role in IVF or ICSI failure. One publication demonstrated that the DNA fragmentation test is a useful tool for male factor evaluation [82]. High DNA fragmentation in the semen can interfere with the fertilization rate, embryo development rate, and implantation rate. Furthermore, it can increase the chance of spontaneous miscarriage [83–85]. The generation of sperm DNA fragmentation is initiated during maturation in the seminiferous tubule [86] and during sperm chromatin packaging in spermiogenesis [86–88].

In spermatozoa with low-level chromatin damage, fertilization capability remains intact due to the self-repairing action of the oocyte [89]. However, with higher chromatin damage, reproductive success depends on the extent of DNA damage and the repairing ability of the oocyte [90]. Young oocytes have a better repairing capacity than older oocytes [91]. In cases of severe sperm chromatin damage beyond repair, the embryo's development might fail to implant or be developmentally delayed [91, 92]. The sperm's DNA damage might not impact the fertilization rate, but the damage of the paternal part can have an effect later (late paternal effect), resulting in delayed embryo development during genomic activation—at the stage of development when there are 4–8 cells [93]—or later, at the time of implantation, leading to implantation failure or miscarriage.

Today's sperm DNA fragmentation tests have a variety of methods. The most commonly used tests in andrology laboratories are the sperm chromatin structural assay (SCSA), terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) assay, sperm chromatin dispersion (SCD) test, and the alkaline Comet test.

#### *Semen Analysis and Infertility DOI: http://dx.doi.org/10.5772/intechopen.107625*

There are variations in clinical thresholds with cut-off level among these tests as well as the sites of the damaged DNA detected [94]. The best assay for DNA fragmentation has not been determined yet. In a systematic review and meta-analysis study, a threshold of 20% of fragmented DNA in the semen sample regarding the SCSA, TUNEL, and SCD tests can be used to differentiate between infertile and fertile men, with a sensitivity of 79% and specificity of 86% [95]. A threshold level of 20–30% of SCSA and SCD tests correlate with the duration of infertility, decreasing the chance of success in intrauterine insemination (IUI), in vitro fertilization (IVF), and intracytoplasmic sperm injecton (ICSI) and increasing the risk of miscarriage [96, 97]. The fragmented DNA is similar to the tip of the iceberg. The hidden part of the fragmented DNA cannot be detected; however, they are prone to be damaged during the process of *in vitro* manipulation of the sperm during assisted reproductive technology.

A DNA fragmentation test behaves the same as semen analysis in that it cannot discriminate between infertile from fertile men, nor can it be used to detect the success in assisted reproductive technology (ART) cycles. The test is not independent in that it still relies on the partner's factors—for example, oocyte quality and age. Basic semen analysis and sperm DNA fragmentation tests complement each other in the diagnosis of male infertility; however, they play a different part in the aspect of male fertility care.

In the clinical setting, sperm DNA fragmentation plays a role in fulfilling the diagnosis of male infertility, especially in the male partner who has specific conditions that require further analysis beyond the basic semen analysis.

#### *4.1.1 Varicocele*

Spermatozoa from a male partner with varicocele have a high potential to be affected by osmotic stress due to high temperature in the testicular environment. These factors result in sperm DNA fragmentation in at least 50% of cases [98]. A systematic review and meta-analysis demonstrated an improved DNA fragmentation rate after varicocelectomy and achievement of pregnancy in comparison to no surgery [99]. After varicocelectomy, a DNA fragmentation test can be a valuable prognostic tool to guide a suitable infertility treatment for a couple. A lower DNA fragmentation than the threshold can indicate a better outcome for natural conception, IVF, and ICSI. The type of treatment depends on the female factor. In the case of persistently high DNA fragmentation, the appropriate treatment can be assisted reproductive technology (ART), either with or without specific sperm selection for better sperm quality [100].

Sperm DNA fragmentation and male infertility are identified in subclinical varicocele. However, the controversial issue related to varicocele is that apparent vein dilation is not found upon examination; it is detected by doppler ultrasound. There was no significant difference in sperm DNA fragmentation rates between fertile and infertile men with subclinical varicocele [101]. A systematic review and meta-analysis study provided evidence that sperm DNA fragmentation rate is comparable between clinical and subclinical varicocele. However, varicocelectomy can only improve the fragmentation rate significantly in clinical varicocele [102].

#### *4.1.2 Idiopathic infertility and unexplained infertility*

Unexplained infertility is responsible for 15–30% of infertile patients. It means they have been investigated using a conventional diagnostic approach for the cause of infertility, but no clear cause of infertility was found [103]. However, about 40–50% of unexplained or idiopathic infertile couples have elevated sperm DNA fragmentation [104]. Likewise, men who have been diagnosed with idiopathic infertility are more likely to have abnormal semen parameters based on basic semen analysis without any obvious abnormality [105]. This information implies that an extended or advanced investigation might be necessary to uncover the causes of infertility in this group [105].

Sperm DNA damage might be one of the main causes of male infertility, especially in cases where infertility is idiopathic or unexplained. The added benefit of the sperm DNA fragmentation evaluation, apart from basic semen analysis, is that it might improve the chance of pregnancy, both natural conception and assisted reproduction that uses adjunctive treatment to improve sperm DNA integrity.

#### *4.1.3 Recurrent pregnancy loss*

The European Society of Human Reproduction and Embryology (ESHRE) has defined the terms of recurrent pregnancy loss as "at least two spontaneous miscarriages starting from natural conception until 24 weeks of gestation" [106]. The sperm DNA fragmentation rate is significantly higher in men with female partners who have had recurrent pregnancy loss than in a fertile female control group having at least one ongoing pregnancy or live birth [107, 108]. The mechanism of sperm fragmented DNA that initiates recurrent pregnancy loss has not been determined yet. However, one hypothesis is that the oocyte repair mechanism might be the main culprit, leading to poor blastocyst development, recurrent implantation failure, and pregnancy loss [108].

#### *4.1.4 Intrauterine insemination*

There is evidence that men with a sperm DNA fragmentation rate diagnosed above 27% by SCSA have higher early pregnancy loss and a lower pregnancy rate than the general infertile population with a lower sperm DNA fragmentation [109, 110]. According to this information, the sperm DNA fragmentation test has an additional benefit of guiding the clinician to choose the treatment of choice for each infertile couple. In case of the sperm DNA fragmentation being higher than the threshold, the couples should have complementary treatment before IUI to ameliorate the sperm DNA fragmentation rate. However, in couples with advanced female age, assisted reproduction should be considered early on due to the oocyte's diminished repair ability and the risk of chromosome abnormality, which are the main causes of treatment failure [94].

#### *4.1.5 Assisted reproduction (in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI)*

High sperm DNA fragmentation impacts the likelihood of successful pregnancy in a natural cycle, intrauterine insemination, and advanced treatments such as IVF and ICSI. There are several studies demonstrating that elevated sperm DNA fragmentation adversely impacts IVF and ICSI. Mainly, it increases spontaneous abortion and recurrent implantation failure. Additionally, it decreases the live birth rate [111–113]. Evidence suggests that the impact of sperm DNA fragmentation might be higher in IVF than in ICSI [114]. It was postulated that the sperm needs to be incubated with

#### *Semen Analysis and Infertility DOI: http://dx.doi.org/10.5772/intechopen.107625*

the oocyte *in vitro* longer in IVF than in ICSI. The duration of exposure of sperm to the external environment during IVF might generate more DNA damage than ICSI, which requires only a short time after ejaculation until successful fertilization [115]. Additionally, the sperm is a significant source of reactive oxygen species. IVF requires the direct contact of at least one hundred thousand sperms and an oocyte for natural fertilization. The exposure of the oocyte to oxidative stress from sperm could adversely impact the embryo's development [116, 117].

Likewise, high sperm DNA fragmentation has an adverse effect that results in these couples having more spontaneous pregnancy losses than couples with low sperm DNA fragmentation [118]. The authors concluded that using a sperm selection method during assisted reproduction to choose sperm with low fragmented DNA could improve the pregnancy rate and decrease the miscarriage rate [118].

The pathophysiologic cause of an increasing rate of spontaneous abortion in high sperm DNA fragmentation in couples undergoing IVF or ICSI is still inconclusive. One proposed mechanism is that the genetic and epigenetic effects of sperm DNA damage could cause mutation or dysregulation of DNA methylation processes that are crucial for embryo development [119–121]. The other proposed mechanism is the oocyte's ability to repair the sperm DNA defect. In oocyte donation cycles, it was found that a good quality oocyte can counteract the defected sperm DNA. However, oocytes from women of advanced maternal age have diminished repair functions compared to young women [122]. Single-stranded DNA breakage is more likely to be repaired than double-stranded DNA breakage [123, 124]. Therefore, the final impact of sperm DNA fragmentation on the pregnancy outcome still relies on the balance of the oocyte repairing system and the extent of sperm DNA damage [90].

Persistent high sperm DNA fragmentation is one of the leading causes of IVF and ICSI failure. Detection of sperm DNA damage is crucial for treatment success. In cases where no causative factor of elevated sperm fragmented DNA is identified, testicular spermatozoa are the suggested method to improve treatment success in ICSI [125–132]. ICSI uses testicular spermatozoa instead of ejaculated spermatozoa, which could be related to the lower sperm DNA damage in the testicular sperm compared to ejaculated sperm that must transit from the epididymis to the male reproductive tract [132–136].

#### *4.1.6 Other risk factors*

Some lifestyle factors can increase sperm DNA fragmentation. Some examples are smoking, drinking, cannabis consumption, exposure to air pollutants, pesticides, polyaromatic hydrocarbons, and fertilizers. Among these factors, smoking has the most impact on sperm DNA fragmentation [137, 138]. Cannabis consumption also impairs sperm DNA integrity [139]. Advanced paternal age above 40 years also reduces the sperm DNA quality [140–142].

Obesity is another common problem that leads to poor sperm quality due to peripheral aromatization from testosterone to estradiol in the subcutaneous fat. The increasing testicular temperature from subpubic fat and the high estradiol levels in obese men might cause hypogonadism and sperm DNA damage [143].

#### *4.1.7 Sperm cryopreservation*

Sperm cryopreservation is a method to preserve male fertility for future use. This method allows men to preserve their semen. Candidates for sperm cryopreservation

include men who have cancer and require chemotherapy or radiation treatments or healthy men who need to preserve semen for future purposes before a vasectomy or after assisted reproduction. This technique is required for sperm donation. However, the process of sperm freezing and thawing can be harmful to sperm quality. It impacts the sperm's motility, viability, and normal morphology. Additionally, it increases osmotic stress on the spermatozoa and leads to sperm DNA damage [144, 145].

The vitrification technique has recently been developed for sperm cryopreservation. It has the potential to reduce sperm DNA damage compared to conventional slow freezing [146, 147]. However, more research is needed.

Sperm DNA fragmentation tests should be done before and after sperm cryopreservation to help improve the method to reduce the impact of cryopreservation on sperm parameters. Moreover, the tests provide the optimal treatment based on sperm quality. The addition of an antioxidant to the sperm freezing media is one technique to protect the sperm from the harsh conditions of the freezing and thawing process by reducing the osmotic stress-induced DNA damage [148].

#### **4.2 Reactive oxygen species measurement**

One of the causes of male infertility and sperm DNA damage is osmotic stress to the spermatozoa. Reactive oxygen species are derived from the metabolism of oxygen. They play a vital role in cellular signaling pathways, sperm maturation processes, and capacitation [149]. Excessive ROS can have a significant detrimental effect on sperm fertility potential [150]. All along the journey initiated from the seminiferous tubule and through the epididymis, *vas deferens,* and finally to the outlet of the male reproductive tract, spermatozoa are potentially assaulted from oxidative stress, which diminishes fertilizing capability and leads to recurrent implantation failure and pregnancy loss. External osmotic stress interferes with the sperm membrane, while internal osmotic stress acts on lipid peroxidation mechanisms, resulting in sperm DNA damage.

Possible mechanisms of oxidative damage to the spermatozoa are sperm membrane and DNA damage, which decrease sperm motility and fertilization ability. These types of damage can also cause poor embryo development, recurrent implantation failure, and early pregnancy loss [84, 151]. Sperm damage by ROS reduces sperm motility, as demonstrated in both conventional and computer-assisted semen analyses [152, 153].

Human semen consists of various kinds of cells, including mature and immature spermatozoa, epithelial cells, and leukocytes. The main sources of ROS in semen are leukocytes (i.e., extrinsic source) and spermatozoa (i.e., intrinsic source). In addition, environmental factors, such as heavy metals, smoking, varicocele, obesity, chronic illness, and genitourinary tract infection are potential sources of ROS, affecting sperm DNA and resulting in DNA fragmentation and damage [154].

Oxidative stress has been demonstrated to be a main factor responsible for male infertility via sperm dysfunction. Oxidative stress on spermatozoa mainly derives from the excessive ROS and inadequate antioxidants to counteract them. Excess ROS can damage sperm by several mechanisms via the oxidative pathway. Approximately 30–40% of infertile men have oxidative stress that causes male infertility [155].

Spermatozoa are unique biological cells in the human body that have limited self-repair capability due to the lack of a cytoplasmic repairing mechanism. This is the most important reason that spermatozoa are vulnerable to internal and external ROS damage [156]. In addition, the membrane of spermatozoa is composed of polyunsaturated fatty acid that is susceptible to oxygen-induced damage and, hence, lipid

#### *Semen Analysis and Infertility DOI: http://dx.doi.org/10.5772/intechopen.107625*

peroxidation. One consequence of lipid peroxidation is damage to the axoneme and midpiece of spermatozoa, resulting in diminished sperm motility [157, 158]. High levels of ROS are detected in at least 25–40% of infertile men [159, 160] Levels of ROS above the semen's antioxidants result in oxidative stress to the sperm [160]. Currently, ROS measurement by chemiluminescence is the most well-described, advanced method of ROS detection in the seminal fluid [161–163].

#### **4.3 Total antioxidant capacity measurement**

Total antioxidant capacity (TAC) is a diagnostic test to measure enzymatic and non-enzymatic kinds of antioxidants in the seminal fluid during a male infertility work up. The value reflects the redox potential an antioxidant has to osmotic stress. There is substantial evidence that the utility of the TAC measurement lies in its ability to detect a lower TAC in infertile men than in a fertile control [164]. The imbalance between osmotic stress and TAC lead to male infertility. TAC is also used to detect who should have antioxidant supplementation before ICSI, especially in case of previously failed ICSI [165]. The role of an antioxidant supplement in male infertility has been reviewed with the potential to lead to a successful pregnancy [166].

#### **4.4 Assessment of the presence of leukocytospermia and hematospermia**

Normal leukocyte production occurs mainly in the epididymis, where they take responsibility for the immunosurveillance of abnormal sperm via phagocytosis. Leukocytes are composed of granulocytes at 50–60%, macrophages at 20–30%, and T-lymphocytes at 2–5% [167]. Leukocytospermia is an excessive amount of leukocytes—more than the threshold, according to the fifth WHO manual—which can impact the sperm quality, as leukocytes are the main source of ROS [154].

The best laboratory guideline for leukocyte assessment in semen is immunohistochemical staining against the various kinds of leukocytes; however, the method is complicated, time-consuming, and not well standardized [168]. The European Association of Urology recommends antibiotic treatment; however, evidence for improving pregnancy outcomes was not demonstrated [169]. Likewise, there is no clear evidence that either antioxidant or antibiotic treatment improves treatment success in infertile men with leukocytospermia [170].

Hematospermia is a term referring to the presence of gross and microscopic examination in the ejaculate. The pathophysiologic causes can be disorders in the ejaculatory ducts, accessory glands, and urethra. Alternatively, it can have iatrogenic causes. The extensive investigations in the case of hematospermia have been welldocumented [171, 172]. There is some evidence of the relationship between hematospermia and male infertility [173, 174]. Red blood cells might be the main source of toxic substances leading to a decline in sperm quality. Hemolysis especially occurs during sperm cryopreservation and thawing [175].

#### **5. Other extended and advanced examination of semen sample parameters**

#### **5.1 Sperm aneuploidy test**

The sperm aneuploidy test is a direct evaluation of sperm chromosome complements that has been used in a couple with recurrent pregnancy loss. The test

evaluates both structural and numerical chromosome abnormality in spermatozoa. A systematic review and meta-analysis demonstrated the increasing incidence of aneuploidy in spermatozoa in cases of recurrent pregnancy loss [176]. The real benefit and implementation of sperm aneuploidy in a routine laboratory for male infertility has yet to be determined, as molecular analysis in miscarriage has revealed that most chromosome aneuploidy occurs during female meiosis, resulting in meiotic non-disjunction [177]. In addition, the final evaluation of chromosome abnormality on the blastocyst can provide both the meiotic and mitotic origin from either oocyte or spermatozoa—with no need to investigate the oocyte or sperm before fertilization.

#### **5.2 Cytokine assessment in the semen**

Infection and inflammation of the male genitourinary tract play an important role in male infertility due to inflammatory mediators and ROS causing damage to the spermatozoa. Any suspicious male genitourinary tract infection should be thoroughly investigated; otherwise, irreversible sperm damage might occur. The biological markers of infection and inflammation in the ejaculate are leukocyte numbers>1 million/ml, granulocyte elastase >280 ng/ml, and proinflammatory cytokines (e.g., interleukin (IL)-6 > 30 pg./ml, IL-8 > 7000 pg./ml) [178]. Cytokine detection might play a potential role and be a sensitive marker of male genitourinary tract infection and inflammation, especially in asymptomatic and silent cases.

#### **5.3 Immature germ cell assessment**

Immature germ cells can be differentiated from leukocytes by Papanicolaou staining. There is no longer a cut-off value of immature germ cells provided in the fifth and sixth WHO manuals, as there is not a sufficient number of studies to confirm the clinical importance of the value. However, elevation of immature germ cell to more than 15% of total sperm in the ejaculate might be significant and indicative of sperm chromatin immaturity [179]. Investigation into the pathophysiologic cause of high shedding of immature germ cells in the ejaculate and the consequences that ensue are not warranted [179].

#### **6. Conclusion**

Currently, semen analysis has become a standard tool for evaluating male infertility and guiding clinicians to provide appropriate treatments for couples. The sperm parameters in the ejaculate are the biological markers of male fertility. A basic semen analysis provides the initial information related to identifying whether a man is fertile or infertile; however, there is no absolute cut-off value for the inability to achieve conception.

In the modern era of assisted reproduction and molecular genetics, new diagnostic techniques reveal the deeply detailed causes of male infertility to improve the treatment outcome. In the modern world, there is more likely to be an association between men's general health and the environment regarding sperm parameters than previously. Having knowledge of the consequences of these factors on sperm parameters can possibly lead to the development of pharmaceutical components or supplements that improve male fertility.

*Semen Analysis and Infertility DOI: http://dx.doi.org/10.5772/intechopen.107625*

### **Author details**

Suchada Mongkolchaipak Chada IVF Fertility Clinic, Chonburi, Thailand

\*Address all correspondence to: doctorsuchada@gmail.com

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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#### **Chapter 3**

## Utilization of a Fertile Chip in Cases of Male Infertility

*Sirin Aydin and Mehmet Eflatun Deniz*

#### **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.

**Keywords:** male infertility, fertile chip, microfluidic sperm preparation, embryo, sperm quality

#### **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 [10–12].

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.

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].

#### **Pituitary Hypothalamic Causes:**


#### **Primary Gonadal Conditions**


#### **Sperm Transport Disorders**


#### **Table 1.**

*Male infertility causes.*

#### *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 headto-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, antisperm 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].


#### **1.2 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 swimup 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).


*Utilization of a Fertile Chip in Cases of Male Infertility DOI: http://dx.doi.org/10.5772/intechopen.107108*

• **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].

**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.

**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

**Figure 1.** *Separation of sperm by zeta potential.*

#### **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).*

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 *Utilization of a Fertile Chip in Cases of Male Infertility DOI: http://dx.doi.org/10.5772/intechopen.107108*

**Figure 3.** *Sperm appearance in the PICSI petri dish.*

autosomal disomy, diploidy, and sex chromosome disomy were significantly lower in HA-linked sperm than in non-binding sperm [69].

**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**).

**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.

**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,

**Figure 4.** *Separation of sperm by electrophoretic method-MicroflowCS-10.*

**Figure 5.** *Sperm selection for IMSI.*

#### *Utilization of a Fertile Chip in Cases of Male Infertility DOI: http://dx.doi.org/10.5772/intechopen.107108*

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

#### **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.*

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].

#### **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 metaanalyses 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.

*Utilization of a Fertile Chip in Cases of Male Infertility DOI: http://dx.doi.org/10.5772/intechopen.107108*

#### **Author details**

Sirin Aydin1 \* and Mehmet Eflatun Deniz<sup>2</sup>

1 Dr. Turgut Noyan Application and Research Center, Baskent University Adana, Turkey

2 Department of Urology, City Hospital, Adana, Turkey

\*Address all correspondence to: dr.sirinaydinn@gmail.com

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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### Section 3
