**Sperm Cell in ART**

## Dejan Ljiljak1, Tamara Tramišak Milaković2, Neda Smiljan Severinski2, Krunoslav Kuna1 and Anđelka Radojčić Badovinac<sup>2</sup> *1University Hospital Center "Sisters of Mercy", Zagreb 2University Hospital Center Rijeka, Rijeka Croatia*

## **1. Introduction**

Infertility today represents a global problem. Male factor contributes in approximately 50% of infertile couples. In the last decade we are witnesses of the decreased quality of semen and the increased frequency of testicular cancer and cryptorchidism. Currently, the assessment of semen quality is based on the routine semen analysis including sperm count, morphology and motility. Although variation and combination among these three main factors articulate few diagnosis, nowadays developed assisted reproduction techniques (ART), especially intracytoplasmatic sperm injection (ICSI) may be used to treat most of the male infertility problems. In general we can say that traditional semen parameters provide a limited degree of diagnostic information, thus we are aware that these indexes of diagnosis should be revisited, which includes more specific test of sperm assessments, such as DNA tests and sperm proteome.

Spermatogenesis is a process that includes physiological, morphological and biochemical changes. After a complex process from the round diploid spermatogonia to haploid spermatozoa, a mature sperm just has an ability to fertilize a mature oocyte. If any errors occur during spermatogenesis process, appropriate sperm will not be produced. Thus, in our ART practice, it is important to understand normal physiology of spermatogenesis and find the reason of abnormal situation. In this chapter we will review today knowledge about:


#### **2. Spermatogenesis**

The sperm cell is composed of a sperm head, a sperm neck and a sperm tail. Whole sperm is covered by the sperm plasma membrane called plasmalemma (Picture 1.). The sperm head

Sperm Cell in ART 67

is composed of a nucleus and an acrosome. The nucleus contains sperm DNA (half number of chromosomes) and the acrosome has important enzymes for fertilization. The sperm neck or midpiece has 100 sperm mitochondria which generate energy for the sperm tail. The sperm tail is based on 9 + 2 microtubules. The microtubule doublets are connected doublet-

A spermatogenesis basically includes the mitotic expansion of stem cells, the meiotic recombination of genetic information and the haploid spermatid production (Picture 2.). The aim of the process is to produce a highly specialized mature sperm cell which can bind to the oocyte. The paternal inherited centrosome is essential for normal fertilization, chromatin packaging and early embryogenesis. The complete matured spermatozoon must undergo acrosome development, nuclear elongation and condensation, the formation of middle piece

The primordial germ cells in fetal testis are enclosed in tubules which form very proliferative active cells called the gonocytes. The meiotic prophase is inhibited. Spermatogonia are positioned on seminiferous tubules and have a connection with the Sertoli cells. The basement membrane and Sertoli cells form the blood-testis barrier. After birth, gonocytes rise to a population of spermatogonia which constitute the stem cell pool. The spermatogonia are characterized by mitotic division and they are inactive until the puberty. The diploid spermatogonia differentiate into primary spermatocytes. The primary spermatocytes undergo first meiotic division and create secondary spermatocytes. After the second meiotic division four haploid spermatids are created. After the morphological differentiation a mature sperm is formed. The Leydig cells are responsible for the production of testosterone which is necessary for spermatogenesis. Some other hormones are also responsible for spermatogenesis. The Luteinising hormone and the follicle stimulating hormone have important influence on right spermatogenesis as well. The first one (FSH) stimulates the Leydig cells to make testosterone and maintain mitotic division. The second one (FSH) is obligated for the influence on Sertoli cells. Sertoli cells produce

to-doublet by dynein arms.

and tail, and the reduction of cytoplasm.

inhibin B. Spermatogenesis process takes about 3 weeks.

chromosomes

Spermatogonium Diploid / 46 Spermatocytogenesis

Spermatid Haploid / 23 Spermiogenesis

Mature sperm cell Haploid / 23 Spermiogenesis

Primary spermatocyte Diploid / 46 Spermatidogenesis (Meiosis I)

Secondary spermatocyte Haploid / 23 Spermatidogenesis (Meiosis II)

Process

Cell Ploidy / number of

Table 1.

Picture 1. Diagram of human spermatozoon

Picture 1. Diagram of human spermatozoon

is composed of a nucleus and an acrosome. The nucleus contains sperm DNA (half number of chromosomes) and the acrosome has important enzymes for fertilization. The sperm neck or midpiece has 100 sperm mitochondria which generate energy for the sperm tail. The sperm tail is based on 9 + 2 microtubules. The microtubule doublets are connected doubletto-doublet by dynein arms.

A spermatogenesis basically includes the mitotic expansion of stem cells, the meiotic recombination of genetic information and the haploid spermatid production (Picture 2.). The aim of the process is to produce a highly specialized mature sperm cell which can bind to the oocyte. The paternal inherited centrosome is essential for normal fertilization, chromatin packaging and early embryogenesis. The complete matured spermatozoon must undergo acrosome development, nuclear elongation and condensation, the formation of middle piece and tail, and the reduction of cytoplasm.

The primordial germ cells in fetal testis are enclosed in tubules which form very proliferative active cells called the gonocytes. The meiotic prophase is inhibited. Spermatogonia are positioned on seminiferous tubules and have a connection with the Sertoli cells. The basement membrane and Sertoli cells form the blood-testis barrier. After birth, gonocytes rise to a population of spermatogonia which constitute the stem cell pool. The spermatogonia are characterized by mitotic division and they are inactive until the puberty. The diploid spermatogonia differentiate into primary spermatocytes. The primary spermatocytes undergo first meiotic division and create secondary spermatocytes. After the second meiotic division four haploid spermatids are created. After the morphological differentiation a mature sperm is formed. The Leydig cells are responsible for the production of testosterone which is necessary for spermatogenesis. Some other hormones are also responsible for spermatogenesis. The Luteinising hormone and the follicle stimulating hormone have important influence on right spermatogenesis as well. The first one (FSH) stimulates the Leydig cells to make testosterone and maintain mitotic division. The second one (FSH) is obligated for the influence on Sertoli cells. Sertoli cells produce inhibin B. Spermatogenesis process takes about 3 weeks.


Table 1.

Sperm Cell in ART 69

The sperm chromatin is very tightly compacted and the result is a paternal genome inactivation. Nuclear proteins and DNA are connected in a unique way. Nuclear remodelling and condensation in the spermatid combine with histone modification and displacement with transition proteins and then by protamines. However, around 15% of sperm DNA remains packaged by histones. The reason lays in the specific manner of oocyte activation after sperm entry. Disulfide cross-links between the cysteine-rich protamines are responsible for the compaction of chromatin and the stabilization of paternal genome. The genome is protected from oxidation or temperature elevation in the female reproductive tract. Human testis express two protamines: protamine 1 (P1) and protamine 2 (P2). Equal amounts of P1 and P2 are considered normal for human spermatozoa. Any unbalance of P1 and P2 ratio is associated with male infertility. Mouse knockout models demonstrate that the sperm protamine haploinsufficiency directly impairs spermatogenesis and embryo development. Through evolution protamines have increased the number of positively charged residues. These positively charged residues create highly condensed complex with

The protamine 1 is synthesized as a mature protein and protamine 2 as a precursor and protamine 1 and 2 differ from each other only by the N-terminal extension of 1-4 residues.

Protamine 2 is zinc-finger protein with one Cys2-His motif and they are expressed only in some mammals. Both, P1 and P2 undergo post-transcriptional modifications before binding to the DNA. After binding, protamines and DNA make highly compact nucleoprotamine complex. Khara et al., (1997) showed first comparison P1/P2 ratio related to IVF and found a P1/P2 ratio between 0.55 and 0.29 in the group with fertilization index (FI) ≥50% and three of the infertile patients who had a F1 below 50% had a ratio outside this range (Khara et al.,

The sperm DNA damage is clearly associated with male infertility. Small part of spermatozoa from fertile men also has detectable levels of DNA damage. Factors that cause the DNA damage include protamine deficiency, apoptosis, chemotherapy, ROS, cigarette smoking and varicoceles. The DNA fragmentation is characterized by single and double strand breaks. Oxidative stress is a result of the production of reactive oxidative species (ROS). Spermatozoa are vulnerable to ROS because they have a small amount of cytoplasm which does not contain antioxidant molecules and repair system. The DNA fragmentation is associated with diminished motility, morphology and sperm count. Also the DNA fragmentation has predictive value for unsuccessful IVF cycle. The most interesting group of patients which go to IVF is idiopathic infertility. Significant paternal contribution to sex chromosome trisomy has been described. Spermatozoa with numerical chromosome abnormalities are able to fertilize oocyte. The increase of sperm aneuploidy rate is associated with lower implantation and pregnancy rate. The sperm mitochondria represent the biggest source of reactive oxygen species. Oxidative stress induces activation of free radicals by the mitochondria which later induce oxidative DNA damage and DNA fragmentation. The

1997). Carrel and Liu (2001) describe the undetectable protamine 2 in infertile males.

**4. DNA damage during spermatogenesis** 

majority of DNA damage is caused by oxidative stress.

**3. Sperm chromatin structure**

DNA.

Picture 2. Spermatogenesis

Picture 2. Spermatogenesis

## **3. Sperm chromatin structure**

The sperm chromatin is very tightly compacted and the result is a paternal genome inactivation. Nuclear proteins and DNA are connected in a unique way. Nuclear remodelling and condensation in the spermatid combine with histone modification and displacement with transition proteins and then by protamines. However, around 15% of sperm DNA remains packaged by histones. The reason lays in the specific manner of oocyte activation after sperm entry. Disulfide cross-links between the cysteine-rich protamines are responsible for the compaction of chromatin and the stabilization of paternal genome. The genome is protected from oxidation or temperature elevation in the female reproductive tract. Human testis express two protamines: protamine 1 (P1) and protamine 2 (P2). Equal amounts of P1 and P2 are considered normal for human spermatozoa. Any unbalance of P1 and P2 ratio is associated with male infertility. Mouse knockout models demonstrate that the sperm protamine haploinsufficiency directly impairs spermatogenesis and embryo development. Through evolution protamines have increased the number of positively charged residues. These positively charged residues create highly condensed complex with DNA.

The protamine 1 is synthesized as a mature protein and protamine 2 as a precursor and protamine 1 and 2 differ from each other only by the N-terminal extension of 1-4 residues.

Protamine 2 is zinc-finger protein with one Cys2-His motif and they are expressed only in some mammals. Both, P1 and P2 undergo post-transcriptional modifications before binding to the DNA. After binding, protamines and DNA make highly compact nucleoprotamine complex. Khara et al., (1997) showed first comparison P1/P2 ratio related to IVF and found a P1/P2 ratio between 0.55 and 0.29 in the group with fertilization index (FI) ≥50% and three of the infertile patients who had a F1 below 50% had a ratio outside this range (Khara et al., 1997). Carrel and Liu (2001) describe the undetectable protamine 2 in infertile males.

#### **4. DNA damage during spermatogenesis**

The sperm DNA damage is clearly associated with male infertility. Small part of spermatozoa from fertile men also has detectable levels of DNA damage. Factors that cause the DNA damage include protamine deficiency, apoptosis, chemotherapy, ROS, cigarette smoking and varicoceles. The DNA fragmentation is characterized by single and double strand breaks. Oxidative stress is a result of the production of reactive oxidative species (ROS). Spermatozoa are vulnerable to ROS because they have a small amount of cytoplasm which does not contain antioxidant molecules and repair system. The DNA fragmentation is associated with diminished motility, morphology and sperm count. Also the DNA fragmentation has predictive value for unsuccessful IVF cycle. The most interesting group of patients which go to IVF is idiopathic infertility. Significant paternal contribution to sex chromosome trisomy has been described. Spermatozoa with numerical chromosome abnormalities are able to fertilize oocyte. The increase of sperm aneuploidy rate is associated with lower implantation and pregnancy rate. The sperm mitochondria represent the biggest source of reactive oxygen species. Oxidative stress induces activation of free radicals by the mitochondria which later induce oxidative DNA damage and DNA fragmentation. The majority of DNA damage is caused by oxidative stress.

Sperm Cell in ART 71

characteristics such as apoptosis, DNA integrity and membrane maturation are not directly targeted by routine sperm preparation techniques. At this moment magnet activated cell sorting (MACS) becomes novel technique for sperm separation based on presence of anexin V as apoptotic marker. Also modified ICSI called PICSI is commonly used for single sperm selection on the level of membrane maturity for sperm binding on hyaluron acid binding sites. Generally, sperm selection can be based on the sperm surface charge (electrophoresisbased technology), non-apoptotic sperm selection, selection based on the sperm membrane maturity and selection based on the sperm ultramorphology. Electrophoresis-based technology separates spermatozoa based on the size and electronegative charge. The externalization of phosphatidylserine (apoptotic marker) allows binding with Annexin-Vconjugated paramagnetic microbeads which separates apoptotic spermatozoa using a

magnetic-activated cell sorting system (MACS, Miltenyi Biotec GmbH, Germany).

replacement reaction. Prog Nucleic Acid Res Mol Biol 1991;40:25-94.

chromatin with folded bull protamine. J Biol Chem 2004;279:20088-20095. [4] Oliva R. Protamines and male infertility. Hum Reprod Update 2006;4:417-435.

invertebrate protamines. Chromosoma 2003;111:473-482.

of eutherian mammals. Mol Reprod Dev 2002;61:519-527.

and embryo death in mice. Biol Reprod 2003;69:211-217.

fragmentation. J Androl 2003;1:59-66.

[1] Oliva R, Dixon GH. Vertebrate protamine genes and the histone-to-protamine

[2] Lewis JD, Song Y, de Jong ME, Bagha SM, Ausio J. A walk though vertebrate and

[3] Vilfan ID, Conwell CC, Hud NV. Formation of native-like mammalian sperm cell

[5] Corzett M, Mazrimas J, Balhorn R. Protamine 1: protamine 2 stoichiometry in the sperm

[6] Aoki VW, Moskovtsev SI, Willis J, Liu L, Mullen JBM, Carrell DT. DNA integrity is compromised in protamine-deficient human sperm. J Androl 2005;26:741-748. [7] Cho C, Jung-Ha H, Willis WD i sur. Protamine 2 deficiency leads to sperm DNA damage

[8] Balhorn R. The protamine family of sperm nuclear proteins. Genome Biology 2007;8:227-

[9] Fernandez JL, Muriel L, Rivero MT, Goyanes V, Vazquez R, Alvarez JG. The sperm chromatin dispersion test: a simple method for the determination of sperm DNA

[10] Larson KL, DeJonge C, Barnes A, Jost L, Evenson DP. Relationship between assisted reproductive techniques (ART) outcome and status of chromatin integrity as measured

[12] Kutchino Y i sur. Misreading of DNA templates containing 8-hydroxydeoxyguanosine

[13] Aoki VW, Carrell DT. Human protamines and the developing spermatid: their structure, function, expression and relationship with male infertility. Asian J Androl

[14] Sakkas D, Alvarez JG. Sperm DNA fragmentation: mechanisms of origin, impact on

by the sperm chromatin structure assay (SCSA). Hum Reprod 2000;15:1717-1722. [11] Zini A, Sigman M. Are tests of sperm DNA damage clinically useful. J Androl

at the modified base and at adjacent residues. Nature 1987;327:77-79.

reproductive outome, and analysis. Fertil Steril 2010;4:1027-1036.

**10. References**

234.

2009;3:219-229.

2003;5:315-324.

## **5. Apoptosis**

The apoptosis represents normal physiological process in spermatogenesis. About 75% potential spermatozoa are destructed by the programmed cell death. The apoptosis acts like selective factor of the early germ cells and prevents overproliferation of germ cells. Also the abnormal sperm formation is excluded from spermatogenesis. Sertoli cells can support a specified number of germ cells. Some spermatozoa with DNA damage or fragmentation escaped apoptosis and exist in the semen. Men with abnormal sperm parameters have higher levels of apoptotic protein Fas. Apoptotic protein Fas is strongly correlated with poor sperm concentration and abnormal sperm morphology. Also some other apoptotic markers can be found in human sperm such Bcl-x, p53, caspase and anexin V.

## **6. Oxidative stress**

Reactive oxygen species (ROS) are the product of the normal metabolism in a cell. Free radicals are highly chemically reactive because of the unpaired electrons. Also ROS are produced by leukocytes in phagocytic process. ROS can effect on sperm DNA integrity. High levels of reactive oxygen species are detected in the semen of infertile men. Reactive oxygen species cause hypercondensation of DNA as a result of the oxidation of sperm protein sulfhydryl groups. The post-testicular genital infection results in the leukocytospermia and the increased levels of DNA damage. ROS can damage the DNA by causing deletions and mutations. Antioxidant therapy has shown a decreasing sperm DNA fragmentation.

## **7. Y chromosome microdeletions**

Microdeletions in the Y chromosome genes are associated with impaired or absent spermatogenesis. Three regions of the Y chromosome azoospermic factor AZFa, AZFb and AZFc are crucial for an adequate process of spermatogenesis. Deletions in AZFa region are associated with Sertoli cells only, deletions in AZFb region with spermatogenic arrest and deletions in AZFc region are associated with the spermatogenic arrest at the spermatid stage. The frequency of Y chromosome microdeletions affects approximately 5-15% infertile men. Previous studies have shown that boys born from oligozoospermic men treated using ICSI have an increased risk of carrying Y chromosome deletions.

## **8. Centrosome**

The centrosome consists of two centrioles in a perpendicular arrangement and pericentriolar material. After the fusion of sperm and oocyte, sperm tail is incorporated into ooplasm and centriolar region forms the sperm aster which acts to guide the female pronucleus towards the male pronucleus. The maternal centrosome is fully degradeted, thus the centrosome of zygote is mainly inherited from the sperm. After fertilization normally formed centriole is an essential proper cell division. The centrosome disfunction may lead to the numerical chromosomal abnormalities. The human sperm centrosome is responsible for normal syngamy and an early embryonic development.

## **9. New techniques of sperm selection in ART**

The routine sperm preparation techniques are density gradient centrifugation and swim-up. They depend on the sedimentation or migration ways to separate spermatozoa. The sperm

The apoptosis represents normal physiological process in spermatogenesis. About 75% potential spermatozoa are destructed by the programmed cell death. The apoptosis acts like selective factor of the early germ cells and prevents overproliferation of germ cells. Also the abnormal sperm formation is excluded from spermatogenesis. Sertoli cells can support a specified number of germ cells. Some spermatozoa with DNA damage or fragmentation escaped apoptosis and exist in the semen. Men with abnormal sperm parameters have higher levels of apoptotic protein Fas. Apoptotic protein Fas is strongly correlated with poor sperm concentration and abnormal sperm morphology. Also some other apoptotic markers

Reactive oxygen species (ROS) are the product of the normal metabolism in a cell. Free radicals are highly chemically reactive because of the unpaired electrons. Also ROS are produced by leukocytes in phagocytic process. ROS can effect on sperm DNA integrity. High levels of reactive oxygen species are detected in the semen of infertile men. Reactive oxygen species cause hypercondensation of DNA as a result of the oxidation of sperm protein sulfhydryl groups. The post-testicular genital infection results in the leukocytospermia and the increased levels of DNA damage. ROS can damage the DNA by causing deletions and mutations.

Microdeletions in the Y chromosome genes are associated with impaired or absent spermatogenesis. Three regions of the Y chromosome azoospermic factor AZFa, AZFb and AZFc are crucial for an adequate process of spermatogenesis. Deletions in AZFa region are associated with Sertoli cells only, deletions in AZFb region with spermatogenic arrest and deletions in AZFc region are associated with the spermatogenic arrest at the spermatid stage. The frequency of Y chromosome microdeletions affects approximately 5-15% infertile men. Previous studies have shown that boys born from oligozoospermic men treated using

The centrosome consists of two centrioles in a perpendicular arrangement and pericentriolar material. After the fusion of sperm and oocyte, sperm tail is incorporated into ooplasm and centriolar region forms the sperm aster which acts to guide the female pronucleus towards the male pronucleus. The maternal centrosome is fully degradeted, thus the centrosome of zygote is mainly inherited from the sperm. After fertilization normally formed centriole is an essential proper cell division. The centrosome disfunction may lead to the numerical chromosomal abnormalities. The human sperm centrosome is responsible for normal

The routine sperm preparation techniques are density gradient centrifugation and swim-up. They depend on the sedimentation or migration ways to separate spermatozoa. The sperm

can be found in human sperm such Bcl-x, p53, caspase and anexin V.

Antioxidant therapy has shown a decreasing sperm DNA fragmentation.

ICSI have an increased risk of carrying Y chromosome deletions.

syngamy and an early embryonic development.

**9. New techniques of sperm selection in ART**

**5. Apoptosis**

**6. Oxidative stress**

**8. Centrosome**

**7. Y chromosome microdeletions**

characteristics such as apoptosis, DNA integrity and membrane maturation are not directly targeted by routine sperm preparation techniques. At this moment magnet activated cell sorting (MACS) becomes novel technique for sperm separation based on presence of anexin V as apoptotic marker. Also modified ICSI called PICSI is commonly used for single sperm selection on the level of membrane maturity for sperm binding on hyaluron acid binding sites. Generally, sperm selection can be based on the sperm surface charge (electrophoresisbased technology), non-apoptotic sperm selection, selection based on the sperm membrane maturity and selection based on the sperm ultramorphology. Electrophoresis-based technology separates spermatozoa based on the size and electronegative charge. The externalization of phosphatidylserine (apoptotic marker) allows binding with Annexin-Vconjugated paramagnetic microbeads which separates apoptotic spermatozoa using a magnetic-activated cell sorting system (MACS, Miltenyi Biotec GmbH, Germany).

#### **10. References**


**6**

*Spain* 

**Meiotic Chromosome Abnormalities and** 

It is generally accepted that infertility affects 15% of couples at reproductive ages. The causes of infertility are 38% female, 20% male, 27% mixed and 15% unknown (Ferlin et al., 2007). The male factor is the sole responsible party or a copartner in infertility in 50% of couples. About 7-8% of men have infertility problems or are the cause of miscarriages. Chromosomal causes rank high among the causes of infertility. 50% of first-trimester abortive eggs have aneuploidy (Hassold et al., 1980). In second and third-trimester miscarriages, the aneuploidy rate drops to 15% and 5%, respectively (Simpson, 2007). Among living newborns, 0.5% to 1% shows aneuploidy (Gardner and Sutherland, 2004). Among the infertile population, 15% of infertility is due to chromosomal or genetic reasons

The prevalence of chromosomal alterations that are only meiotic, with a normal mitotic karyotype, is unknown. Checking for sperm chromosomal alterations requires testicular biopsy, which is an invasive procedure because meiotic cells, spermatocytes I and II present in ejaculate does not tend to be valid, due to their scarcity and poor condition. Unlike mitosis, which can affect other organs and functions, chromosomal alterations of solely meiosis may have an impact on reproductive capacity. Meiotic chromosomal anomalies can cause alterations to one or more of the basic seminal parameters including sperm count, motility and morphology, even lead to the formation of aneuploid gametes. At a clinical level, men with meiotic anomalies will have primary or secondary infertility or produce gestations with miscarriages. Secondary infertility or difficulty in having another child can be explained by the coexistence of altered cell lines and other normal cell lines (mosaicism). The meiotic chromosomal anomalies may be studied by directly observing meiotic cells obtained from testicular biopsies. Understanding impact on fertility requires: 1) ascertaining the patient's reproductive history with his current and past partners, if there were any; 2) semen analysis, which can be normal with regard to its three basic parameters, have somewhat severe alterations in some or all sperm parameters, or even azoospermia; 3) studying the testicular histopathology that, at an optical level, can swing between complete blockage at the level of spermatocyte I or II, through apparently normal spermatogenesis

**1. Introduction** 

(Griffin and Finch, 2005).

and 4) studying aneuploidy present in sperm.

**Spermatic FISH in Infertile Patients**

*Instituto de Reproducción CEFER. ANACER member, Barcelona* 

Simón Marina, Susana Egozcue, David Marina,

**with Normal Karyotype** 

Ruth Alcolea and Fernando Marina


http://www.embryology.ch/anglais/cgametogen/spermato03.html

[20] Tamer MS, Land JA. Effects of advanced selection methods on sperm quality and ART outcome: a systematic review. Hum Rep Update 2011; 719-733.
