Until the 1990s, the majority of cases of severe male factor subfertility were virtually untreatable, however, the introduction of ICSI revolutionized the treatment of male infertility (Palermo et al., 1992). However, ICSI is a subject of an ongoing debate regarding its indications and safety (Govaerts et al., 1996; Griffin et al., 2003; Kurinczuk 2003; Verpoest and Tournaye 2006; Varghese et al., 2007). One of the causes to this is that ICSI must be seen as a symptomatic treatment, not taking the underlying causes of infertility into account.

**2. Male infertility**

O'Flynn O'Brien et al., 2010).

**2.1 Diagnosis and treatment of male infertility**

With a volume 40 times less than that of a somatic cell nucleus the genetic material of a spermatozoon is more compact packaged than in the nucleus of a somatic cell (Ward et al., 1991). During spermiogenesis histones are replaced by the more basic and small protamines (Fuentes-Mascorro et al. 2000). Each unit of mammalian sperm chromatin is a toroid containing 50–60 kb of DNA and individual toroids represent DNA loop-domains highly condensed by protamines and fixed at the nuclear matrix. The toroids are bound by disulfide crosslinks, formed by oxidation of sulfhydryl groups of cysteine present in the protamines and each chromosome represents a garland of toroids (Fuentes-Mascorro et al., 2000; Ward et al., 1993). While in most other species the protamines comprise as much as 95%, human protamines comprise 85% of the spermatozoal nucleoproteins. (Fuentes-Mascorro et al., 2000). This may explain why human sperm chromatin is less compacted and more frequently contains DNA breaks (Bench et al., 1993) compared to other species.

#### **4. Sperm DNA damage**

Human sperm DNA is often not so well packaged as meant to be (Sakkas et al., 1999a) and is therefore susceptible to DNA damage (Irvine et al., 2000). Whilst the mature sperm is a repair-deficient cell (Sega et al., 1978), oocytes and embryos are, to a certain degree, able to repair DNA damage (Matsuda and Tobari 1988; Ahmadi and Ng 1999b).

Sperm possess a variety of abnormalities at the nuclear level and that these anomalies can have an impact on fertility (Evenson et al., 1980; Hewitson 1999; Huszar 1999). The most common types of DNA damage include chemical modification of a base, inter- and intrastrand crosslinks, and single or double DNA strand breaks (Marchetti and Wyrobek 2005).

The origin of human sperm DNA damage is involving both testicular and post-testicular mechanisms. Testicular mechanisms include a) alterations in chromatin modelling during the process of spermiogenesis, and b) abortive apoptosis, whereas post-testicular factors are mostly related to the action of c) reactive oxygen species (ROS), and d) activation of caspases and endonucleases( Reviewed in (Aitken and De Iuliis, 2010 and Sakkas and Alvarez, 2010)).

Oxidative stress during sperm transport through the male reproductive tract is likely the most frequent cause of sperm DNA damage (Aitken and De Iuliis, 2010; Sakkas and Alvarez, 2010). Under normal conditions ROS are necessary for the functioning of sperm (reviewed by Aitken 2006), however, oxidative stress resulting from an over production or reduced antioxidant protection are thought to cause DNA damage (Aitken et al. 1998).

The risk of having ROS induced DNA damaged sperm increases by advanced age, abstinence time, influence of cancer treatment, varicocele and obesity (reviewed in Erenpreiss et al., 2006a; Aitken and De Iulius 2007). Moreover, several studies have reported a negative effect of cigarette smoking on sperm DNA, but data are not conclusive (Robbins et al., 1997; Sun et al., 1997; Rubes et al., 1998; Potts et al., 1999; Saleh et al., 2002b; Sepaniak et al., 2006). Other sources of ROS include organophosphorous pesticides (Sanchez-Pena et al., 2004) and other types of air pollution (Rubes et al., 1998; Selevan et al., 2000; Evenson and Wixon 2005). These agents possess estrogenic properties that are capable of inducing ROS production (Sanchez-Pena et al., 2004; Baker and Aitken 2005; Bennetts et al., 2008).

Role of Sperm DNA Integrity in Fertility 27

The terminal deoxynucleotidyl transferase (TdT)-mediated 2´-deoxyuridine 5´-triphosphatenick end labelling (TUNEL) assay can be applied for both light microscopy and flow cytometry. The assay uses TdT to label the 3'-OH ends of double-stranded DNA breaks, but also works on the single strand 3'-OH (Gorczyka et al., 1993). The assay detects the DNA breaks directly, without any initial step of denaturation as in SCSA or by introducing acid or alkaline pH as in COMET assay. Whilst TUNEL assay based on light microscopy normally

The Sperm chromatin dispersion (SCD) test is a relatively simple test that can be applied either by fluorescence microscopy or by bright field microscopy. The test is based on the principle that sperm with fragmented DNA fails to produce the characteristic halo of dispersed DNA loops that are observed on sperm with non-fragmented DNA, when mixed with aqueous agarose following acid denaturation and removal of chromatin nuclear proteins (Fernandez et al., 2003). In the SCD test normally 500 spermatozoa are analysed.

The WHO semen analysis is a golden standard in diagnosis of male infertility, this despite an intra-individual variation reported to be as high as up to 54% (Keel 2006). The first SCSA studies demonstrated that sperm chromatin parameters varied less within a man (Evenson et al., 1991; Spanò et al., 1998). In a study of 45 men who delivered monthly semen samples Evenson reported an average within-donor CV of the SCSA-parameter DFI around 23% (Evenson et al., 1991). These results were confirmed by another SCSA study of 277 men whose semen was measured two times during a period of six months (Spanò et al., 1998). Also Smit and co-workers in 100 men from an outpatient andrology clinic demonstrated a lower biological variation of sperm DNA fragmentation than the classical WHO sperm parameters (Smit et al., 2007). However, conflicting results were obtained in 282 patients undergoing ART with repeated (between 2 and 5) SCSA measurements. In this study, CV of DFI was as high as 29% (Erenpreiss et al., 2006b). In a more recent study these results were reproduced by Olechuk et al., (2011) who also found the mean CV for DFI to be around 30%. In this study 85% of the men that repeated their SCSA analysis remained in the same DFI

It is evident that infertile men possess more sperm with DNA damage than fertile men (Evenson et al., 1999; Gandini et al., 2000; Host et al., 2000b; Irvine et al., 2000; Larson et al., 2000; Spanò et al., 2000; Carrell and Liu 2001; Hammadeh et al., 2001; Zini et al., 2001a; Sakkas et al., 2002; Saleh et al., 2002b; Zini et al., 2002; Erenpreisa et al., 2003; Muratori et al., 2003; Saleh et al., 2003a). However, very few infertile men are offered a sperm DNA integrity analysis during their fertility work-up, and are therefore not aware of the problem. This despite the fact that in 10-25% of men diagnosed with unexplained infertility sperm DNA damage can, at least partly explain their childlessness (Bungum et al., 2007; Erenpreiss

assess 2-500 cells the flow cytometry TUNEL assess 5-10 000 cells.

category (DFI <30% or DFI >30%) from sample one to sample two.

**7. Sperm chromatin damage and male infertility**

et al.,2008; Smit et al., 2010; Giwercman et al., 2010).

**5.4 Sperm Chromatin Dispersion (SCD) test**

**6. Intra-individual variation of DFI**

**5.3 TUNEL assay**

#### **5. Sperm DNA integrity testing**

Several tests developed to assess sperm DNA damage are available. The most frequently used is the Sperm Chromatin Structure assay (SCSA), the single-cell gel electrophoresis assay (COMET assay) in its alkaline, neutral, 2-tailed versions (Singh et al., 1997; Lewis and Agbaje, 2008; Enciso et al., 2009), the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay (Gorczyka et al., 1993) and the sperm chromatin dispersion (SCD) test (Fernandez et al., 2003). Although these tests correlate to each other (Aravindan et al. 1997; Zini et al. 2001; Perera et al. 2002; Erenpreiss et al. 2004, Donelly et al., 2000) and all measure single and double strand breaks, the methodologies are based on different principles and different aspects of sperm DNA damage (Makhlouf and Niederberger, 2006).

#### **5.1 Sperm Chromatin Structure Assay (SCSA)**

The SCSA® is a flow-cytrometic test based on the fact that damaged sperm chromatin denatures when exposed to a low pH-buffer, whereas normal chromatin remains stable (Evenson et al., 1980). The SCSA measures the denaturation of sperm DNA stained with acridine orange, which differentially stains double- and single stranded DNA. Five to ten thousand cells are analysed. Thereafter, the flow cytometric data is further analyzed using dedicated software (SCSASoft; SCSA Diagnostics, Brookings, SD, USA). Data appears in histo- and cytograms and results given as DAN fragmentation index (DFI) and High DNA stainability (HDS). It is still unclear which mechanisms and types of DNA damage that are lying behind DFI and HDS, however, it is believed that DFI are related to the percentage of sperm with both single strand breaks (SSB) and double strand breaks (DSB) or problems in the histone to protamine exchange. HDS is thought to represent immature sperm. The clear advantage of SCSA is the objectivity of the test as well as the high reproducibility (Giwercman et al., 2003) when ran after the standardised protocol (Evenson et al., 2002). Moreover, the clear cut-off levels in relation to fertility is maybe the most obvious benefit compared to other sperm DNA integrity tests (Bungum et al., 2007). A disadvantage is that an expensive flow cytometer is required to run the analysis. Moreover, the test irreversibly damage spermatozoa; after analysis they cannot be used for fertilisation purposes.

#### **5.2 COMET assay**

The COMET assay (Singh et al., 1997) is a single cell gel electrophoresis of immobilised sperm, which involves their encapsulation in agarose, lysis and electrophoresis. When the electric field is applied the negatively charged DNA will be drawn towards the positively charged anode. While undamaged DNA are too large and will remain in nucleus, the smaller broken DNA fragments move in a given period of time. The amount of DNA that leaves the nucleus is a measure of the DNA damage in the cell. The sperm are stained with a DNA-binding dye and the intensity of the fluorescence is measured by image analysis. The overall structure resembles a COMET with a circular head corresponding to the undamaged DNA that remains in the nucleus and a tail of damaged DNA. COMET assay can be ran under neutral or alkaline conditions. Under neutral conditions (pH 8–9), mainly DSB are detected (Collins et al. 2004). Under alkaline conditions, DSB and SSB (at pH 12.3) and additionally alkali labile sites (at pH≥13) can be visualised resulting in increased DNA migration in the electrophoretic field (Fairbairn et al., 1994). In COMET assay, normally only around 100 cells are analysed.

#### **5.3 TUNEL assay**

26 Embryology – Updates and Highlights on Classic Topics

Several tests developed to assess sperm DNA damage are available. The most frequently used is the Sperm Chromatin Structure assay (SCSA), the single-cell gel electrophoresis assay (COMET assay) in its alkaline, neutral, 2-tailed versions (Singh et al., 1997; Lewis and Agbaje, 2008; Enciso et al., 2009), the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay (Gorczyka et al., 1993) and the sperm chromatin dispersion (SCD) test (Fernandez et al., 2003). Although these tests correlate to each other (Aravindan et al. 1997; Zini et al. 2001; Perera et al. 2002; Erenpreiss et al. 2004, Donelly et al., 2000) and all measure single and double strand breaks, the methodologies are based on different principles and different aspects of sperm DNA damage (Makhlouf and Niederberger, 2006).

The SCSA® is a flow-cytrometic test based on the fact that damaged sperm chromatin denatures when exposed to a low pH-buffer, whereas normal chromatin remains stable (Evenson et al., 1980). The SCSA measures the denaturation of sperm DNA stained with acridine orange, which differentially stains double- and single stranded DNA. Five to ten thousand cells are analysed. Thereafter, the flow cytometric data is further analyzed using dedicated software (SCSASoft; SCSA Diagnostics, Brookings, SD, USA). Data appears in histo- and cytograms and results given as DAN fragmentation index (DFI) and High DNA stainability (HDS). It is still unclear which mechanisms and types of DNA damage that are lying behind DFI and HDS, however, it is believed that DFI are related to the percentage of sperm with both single strand breaks (SSB) and double strand breaks (DSB) or problems in the histone to protamine exchange. HDS is thought to represent immature sperm. The clear advantage of SCSA is the objectivity of the test as well as the high reproducibility (Giwercman et al., 2003) when ran after the standardised protocol (Evenson et al., 2002). Moreover, the clear cut-off levels in relation to fertility is maybe the most obvious benefit compared to other sperm DNA integrity tests (Bungum et al., 2007). A disadvantage is that an expensive flow cytometer is required to run the analysis. Moreover, the test irreversibly

damage spermatozoa; after analysis they cannot be used for fertilisation purposes.

The COMET assay (Singh et al., 1997) is a single cell gel electrophoresis of immobilised sperm, which involves their encapsulation in agarose, lysis and electrophoresis. When the electric field is applied the negatively charged DNA will be drawn towards the positively charged anode. While undamaged DNA are too large and will remain in nucleus, the smaller broken DNA fragments move in a given period of time. The amount of DNA that leaves the nucleus is a measure of the DNA damage in the cell. The sperm are stained with a DNA-binding dye and the intensity of the fluorescence is measured by image analysis. The overall structure resembles a COMET with a circular head corresponding to the undamaged DNA that remains in the nucleus and a tail of damaged DNA. COMET assay can be ran under neutral or alkaline conditions. Under neutral conditions (pH 8–9), mainly DSB are detected (Collins et al. 2004). Under alkaline conditions, DSB and SSB (at pH 12.3) and additionally alkali labile sites (at pH≥13) can be visualised resulting in increased DNA migration in the electrophoretic field (Fairbairn et al., 1994). In COMET assay, normally only

**5. Sperm DNA integrity testing**

**5.1 Sperm Chromatin Structure Assay (SCSA)**

**5.2 COMET assay**

around 100 cells are analysed.

The terminal deoxynucleotidyl transferase (TdT)-mediated 2´-deoxyuridine 5´-triphosphatenick end labelling (TUNEL) assay can be applied for both light microscopy and flow cytometry. The assay uses TdT to label the 3'-OH ends of double-stranded DNA breaks, but also works on the single strand 3'-OH (Gorczyka et al., 1993). The assay detects the DNA breaks directly, without any initial step of denaturation as in SCSA or by introducing acid or alkaline pH as in COMET assay. Whilst TUNEL assay based on light microscopy normally assess 2-500 cells the flow cytometry TUNEL assess 5-10 000 cells.

#### **5.4 Sperm Chromatin Dispersion (SCD) test**

The Sperm chromatin dispersion (SCD) test is a relatively simple test that can be applied either by fluorescence microscopy or by bright field microscopy. The test is based on the principle that sperm with fragmented DNA fails to produce the characteristic halo of dispersed DNA loops that are observed on sperm with non-fragmented DNA, when mixed with aqueous agarose following acid denaturation and removal of chromatin nuclear proteins (Fernandez et al., 2003). In the SCD test normally 500 spermatozoa are analysed.

#### **6. Intra-individual variation of DFI**

The WHO semen analysis is a golden standard in diagnosis of male infertility, this despite an intra-individual variation reported to be as high as up to 54% (Keel 2006). The first SCSA studies demonstrated that sperm chromatin parameters varied less within a man (Evenson et al., 1991; Spanò et al., 1998). In a study of 45 men who delivered monthly semen samples Evenson reported an average within-donor CV of the SCSA-parameter DFI around 23% (Evenson et al., 1991). These results were confirmed by another SCSA study of 277 men whose semen was measured two times during a period of six months (Spanò et al., 1998). Also Smit and co-workers in 100 men from an outpatient andrology clinic demonstrated a lower biological variation of sperm DNA fragmentation than the classical WHO sperm parameters (Smit et al., 2007). However, conflicting results were obtained in 282 patients undergoing ART with repeated (between 2 and 5) SCSA measurements. In this study, CV of DFI was as high as 29% (Erenpreiss et al., 2006b). In a more recent study these results were reproduced by Olechuk et al., (2011) who also found the mean CV for DFI to be around 30%. In this study 85% of the men that repeated their SCSA analysis remained in the same DFI category (DFI <30% or DFI >30%) from sample one to sample two.

#### **7.  However, very few infertile men are offered a sperm DNA integrity analysis during their fertility work-up, and are therefore not aware of the problem. This despite the fact that in 10-25% of men diagnosed with unexplained infertility sperm DNA damage can, at least partly explain their childlessness (Bungum et al., 2007; Erenpreiss et al.,2008; Smit et al., 2010; Giwercman et al., 2010).

Role of Sperm DNA Integrity in Fertility 29

IVF and 223 ICSI cycles were published (Bungum et al., 2007). We observed no statistically significant differences between the outcomes of ICSI and ICSI when a DFI level of 30% was used.. However, in the DFI >30% group, the results of ICSI were significantly better than those of IVF. When comparing ICSI to IVF the odds ratios (ORs) for biochemical pregnancy, clinical pregnancy and delivery were 3.0 (95% CI: 1.4–6.2), 2.3 (5% CI: 1.1–4.6) and 2.2 (95% CI: 1.0–4.5), respectively. Also smaller reports using TUNEL or COMET assays shows that sperm DNA damage is more predictive in IVF than in ICSI (Hammadeh et al., 1998; Host et al., 2000; Simon et al., 2011). In contrast, one single SCSA study has reported that DFI threshold did not predict IVF outcome (Payne et al., 2005). However in this study the

In a mouse model Ahmadi and Ng demonstrated that spermatozoa with DNA damage were able to fertilise oocytes (Ahmadi and Ng 1999a). They also reported sperm DNA damage to be related to poor embryo development (Ahmadi and Ng 1999b), although the oocyte, to a certain degree, was able to repair the sperm DNA damage. The human data from ART populations regarding fertilisation and embryo development in relation to DNA damage is,

In our large study of 611 IVF and ICSI couples we compared fertilisation rates between those having a DFI>30% and those having a DFI≤30%, however, no statistically significant differences were seen, neither for IVF nor for ICSI patients (Bungum et al., 2007).  In contrast, a negative correlation between sperm DNA fragmentation and IVF and ICSI fertilisation rates were reported by others (Sun et al., 1997; Lopes et al., 1998a; Saleh et al., 2003a; Payne et al., 2005). A Danish study (Host et al., 2000a) assessed sperm DNA breaks with the TUNEL- assay in infertile couples and found negative correlations between the proportion of spermatozoa with DNA

In our ART-study from 2007 (Bungum et al., 2007), embryo development was compared between those having a SCSA-DFI>30% and those having a DFI≤30%, however, no statistically significant differences between the groups were seen. Several other authors have reported identical results (Larson et al., 2000; Larson-Cook et al., 2003; Gandini et al., 2004; Boe-Hansen et al., 2006). This, however, is contrasted by the findings of others (Sun et al., 1997; Morris et al., 2002; Saleh et al., 2003a), who showed that DFI levels were negatively correlated with embryo quality after IVF and ICSI. Seli et al. (2004) and Virro et al. (2004) reported that men with a high DFI had higher risk of low blastocyst formation rate

Traditionally, pregnancy loss has been explained by either genetic, structural, infective, endocrine or unexplained causes (reviewed in (Rai and Regan 2006)). However, recently

damage and fertilisation in all groups except for those treated with ICSI.

authors did not discriminate between IVF and ICSI.

**10.2 Fertilisation and embryo development**

however, conflicting.

compared to those with a low DFI.

**10.3 DFI and risk of pregnancy loss**

#### **8. Predictive role of DFI in spontaneous pregnancy**

Only few studies have studied the role of sperm DNA integrity in relation to fertility in an unselected population. The US Georgetown study included 165 couples (Evenson et al., 1991) and the Danish first pregnancy planners study (Spanò et al., 2000) included 215 couples who tried to obtain pregnancy. Both studies analysed sperm DNA damage by the use of SCSA and demonstrated that the chance of spontaneous pregnancy, measured by the time-to-pregnancy (TTP), decreased when DFI, exceeded 20–30% and became infinite when DFI was more than 30%.

These results have been confirmed in a more recent SCSA case-control study of 127 men from infertile couples where female factors were excluded and 137 men with proven fertility. The risk of being infertile was increased when DFI rised above 20% in men with normal standard semen parameters (OR 5.1), whereas if one of the WHO parameters were abnormal, the OR for infertility was increased already at DFI above 10% (OR 16) (Giwercman et al., 2010). This above mentioned study demonstrated that SCSA can be used in prediction of the chance of spontaneous pregnancy, independently of the standard sperm parameters but also that combining WHO parameters with DFI can be beneficial. Giwercman and co-workers claimed that since a DFI >20% was found in 40% of men with otherwise normal standard sperm parameters, in almost half of the cases of unexplained infertility, sperm DNA defects are a contributing factor to the problem.

#### **9. Predictive role of DFI in intrauterine insemination (IUI)**

Several reports have studied DFI in prediction of fertility following intrauterine insemination (IUI). The first report used the TUNEL assay on prepared semen for sperm DNA integrity analysis (Duran et al., 2002). In 154 couples they found lack of pregnancy when DFI was above 12%. Other smaller SCSA studies have confirmed this (Saleh et al., 2003; Boe-Hanssen el al., 2006). In 2007, our group published a study based on 387 IUI cycles where DFI was assessed by SCSA (Bungum et al., 2007). DFI was shown to be a predictor of fertility independent of other sperm parameters. In men having a DFI level below 30% the proportion of children born per cycle was 19.0%. This was in contrast to those having a DFI value above 30% who only had a take-home-baby rate of 1.5 %. The chance of IUI pregnancy started to decrease already when the DFI value exceeded the 20%-level, but became close to zero when exceeding 30%.

#### **10. Predictive role of DFI in IVF and ICSI**

More contrasting data exist regarding role of sperm DNA damage in relation to fertilisation, embryo development and pregnancy outcome in IVF and ICSI.

#### **10.1 IVF and ICSI pregnancy**

The first SCSA studies based on a relatively limited number of couples indicated that DFI above 27% could be used as a cut-off value for infertility (Larsson et al., 2000; Larsson-Cook et al., 2003). However, in 2004 three independent SCSA reports demonstrated that one through the use of IVF and ICSI were able to compensate for poor sperm chromatin quality (Bungum et al., 2004; Virro et al., 2004; Gandini et al., 2004). Then in 2007 data based on 388

Only few studies have studied the role of sperm DNA integrity in relation to fertility in an unselected population. The US Georgetown study included 165 couples (Evenson et al., 1991) and the Danish first pregnancy planners study (Spanò et al., 2000) included 215 couples who tried to obtain pregnancy. Both studies analysed sperm DNA damage by the use of SCSA and demonstrated that the chance of spontaneous pregnancy, measured by the time-to-pregnancy (TTP), decreased when DFI, exceeded 20–30% and became infinite when

These results have been confirmed in a more recent SCSA case-control study of 127 men from infertile couples where female factors were excluded and 137 men with proven fertility. The risk of being infertile was increased when DFI rised above 20% in men with normal standard semen parameters (OR 5.1), whereas if one of the WHO parameters were abnormal, the OR for infertility was increased already at DFI above 10% (OR 16) (Giwercman et al., 2010). This above mentioned study demonstrated that SCSA can be used in prediction of the chance of spontaneous pregnancy, independently of the standard sperm parameters but also that combining WHO parameters with DFI can be beneficial. Giwercman and co-workers claimed that since a DFI >20% was found in 40% of men with otherwise normal standard sperm parameters, in almost half of the cases of unexplained

Several reports have studied DFI in prediction of fertility following intrauterine insemination (IUI). The first report used the TUNEL assay on prepared semen for sperm DNA integrity analysis (Duran et al., 2002). In 154 couples they found lack of pregnancy when DFI was above 12%. Other smaller SCSA studies have confirmed this (Saleh et al., 2003; Boe-Hanssen el al., 2006). In 2007, our group published a study based on 387 IUI cycles where DFI was assessed by SCSA (Bungum et al., 2007). DFI was shown to be a predictor of fertility independent of other sperm parameters. In men having a DFI level below 30% the proportion of children born per cycle was 19.0%. This was in contrast to those having a DFI value above 30% who only had a take-home-baby rate of 1.5 %. The chance of IUI pregnancy started to decrease already when the DFI value exceeded the 20%-level, but became close to

More contrasting data exist regarding role of sperm DNA damage in relation to fertilisation,

The first SCSA studies based on a relatively limited number of couples indicated that DFI above 27% could be used as a cut-off value for infertility (Larsson et al., 2000; Larsson-Cook et al., 2003). However, in 2004 three independent SCSA reports demonstrated that one through the use of IVF and ICSI were able to compensate for poor sperm chromatin quality (Bungum et al., 2004; Virro et al., 2004; Gandini et al., 2004). Then in 2007 data based on 388

**8. Predictive role of DFI in spontaneous pregnancy**

infertility, sperm DNA defects are a contributing factor to the problem.

**9. Predictive role of DFI in intrauterine insemination (IUI)**

DFI was more than 30%.

zero when exceeding 30%.

**10.1 IVF and ICSI pregnancy**

**10. Predictive role of DFI in IVF and ICSI**

embryo development and pregnancy outcome in IVF and ICSI.

IVF and 223 ICSI cycles were published (Bungum et al., 2007). We observed no statistically significant differences between the outcomes of ICSI and ICSI when a DFI level of 30% was used.. However, in the DFI >30% group, the results of ICSI were significantly better than those of IVF. When comparing ICSI to IVF the odds ratios (ORs) for biochemical pregnancy, clinical pregnancy and delivery were 3.0 (95% CI: 1.4–6.2), 2.3 (5% CI: 1.1–4.6) and 2.2 (95% CI: 1.0–4.5), respectively. Also smaller reports using TUNEL or COMET assays shows that sperm DNA damage is more predictive in IVF than in ICSI (Hammadeh et al., 1998; Host et al., 2000; Simon et al., 2011). In contrast, one single SCSA study has reported that DFI threshold did not predict IVF outcome (Payne et al., 2005). However in this study the authors did not discriminate between IVF and ICSI.

#### **10.2 Fertilisation and embryo development**

In a mouse model Ahmadi and Ng demonstrated that spermatozoa with DNA damage were able to fertilise oocytes (Ahmadi and Ng 1999a). They also reported sperm DNA damage to be related to poor embryo development (Ahmadi and Ng 1999b), although the oocyte, to a certain degree, was able to repair the sperm DNA damage. The human data from ART populations regarding fertilisation and embryo development in relation to DNA damage is, however, conflicting.

In our large study of 611 IVF and ICSI couples we compared fertilisation rates between those having a DFI>30% and those having a DFI≤30%, however, no statistically significant differences were seen, neither for IVF nor for ICSI patients (Bungum et al., 2007).  In contrast, a negative correlation between sperm DNA fragmentation and IVF and ICSI fertilisation rates were reported by others (Sun et al., 1997; Lopes et al., 1998a; Saleh et al., 2003a; Payne et al., 2005). A Danish study (Host et al., 2000a) assessed sperm DNA breaks with the TUNEL- assay in infertile couples and found negative correlations between the proportion of spermatozoa with DNA damage and fertilisation in all groups except for those treated with ICSI.

In our ART-study from 2007 (Bungum et al., 2007), embryo development was compared between those having a SCSA-DFI>30% and those having a DFI≤30%, however, no statistically significant differences between the groups were seen. Several other authors have reported identical results (Larson et al., 2000; Larson-Cook et al., 2003; Gandini et al., 2004; Boe-Hansen et al., 2006). This, however, is contrasted by the findings of others (Sun et al., 1997; Morris et al., 2002; Saleh et al., 2003a), who showed that DFI levels were negatively correlated with embryo quality after IVF and ICSI. Seli et al. (2004) and Virro et al. (2004) reported that men with a high DFI had higher risk of low blastocyst formation rate compared to those with a low DFI.

#### **10.3 DFI and risk of pregnancy loss**

Traditionally, pregnancy loss has been explained by either genetic, structural, infective, endocrine or unexplained causes (reviewed in (Rai and Regan 2006)). However, recently

Role of Sperm DNA Integrity in Fertility 31

recommended for a clinical sperm DNA damage evaluation. The normality ranges and

It seems clear that ART, especially ICSI, are able to overcome the natural barriers of sperm DNA damage levels not compatible with fertilisation under natural circumstances. The consequences of this for the progeny are still not clear, however, it is also reason to concern regarding possible consequences of achieving a pregnancy using spermatozoa possessing DNA damage. So far no proven treatment of sperm DNA damage is available. Adequately powered, placebo-controlled trials of antioxidants in the prevention of sperm oxidative

Agarwal, A., Desai,. NR., Ruffoli, R. & Carpi A. (2008). Lifestyle and testicular dysfunction: a

Ahmadi, A. & Ng, S. C. (1999a). Fertilizing ability of DNA-damaged spermatozoa. J Exp

Ahmadi, A. & Ng, S. C. (1999b). Developmental capacity of damaged spermatozoa. Hum

Aitken, J. & De Iulius, G. N. (2007). Origins and concequences of DNA damage in male germ

Aitken, R. J. & De Iuliis, G.N. (2010). On the possible origins of DNA damage in human

Aitken, R. J. (2006). Sperm function tests and fertility. Int J Androl 29, 69-75; discussion 105-

Aitken, R. J. , De Iuliis, G.N. & McLachlan R.I. (2009). Biological and clinical significance of

Aravindan, G. R., Bjordahl, J., Jost, L. K. & Evenson, D. P. (1997). Susceptibility of human

Auger, J., Eustache, F., Andersen, A. G., Irvine, D. S., Jorgensen, N., Skakkebaek, N. E.,

of pregnant women from four European cities. Hum Reprod 16, 2710-2717. Baker, M. A. & Aitken, R. J. (2005). Reactive oxygen species in spermatozoa: methods for

Bench, G.S., Friz, A.M., Corzett, M.H., Morse, D.H. & Balhorn R. (1996). DNA and total

Bennetts, L. E., De Iuliis, G. N., Nixon, B., Kime, M., Zelski, K., McVicar, C. M., Lewis, S. E. &

sperm to in situ DNA denaturation is strongly correlated with DNA strand breaks

Suominen, J., Toppari, J., Vierula, M. & Jouannet, P. (2001). Sperm morphological defects related to environment, lifestyle and medical history of 1001 male partners

monitoring and significance for the origins of genetic disease and infertility.

protamine masses in individual sperm from fertile mammalian subjects. Cytometry

Aitken, R. J. (2008). Impact of estrogenic compounds on DNA integrity in human spermatozoa: Evidence for cross-linking and redox cycling activities. Mutat Res

DNA damage in the male germ line. Int J Androl 32:46-56.

identified by single-cell electrophoresis. Exp Cell Res 236, 231-237

brief update. Biomed Pharmacother 62(8):550-553.

cells. Reprod Biomed Online 14, 727-733

spermatozoa. Mol Hum Reprod 16:3-13.

Reprod Biol Endocrinol 3, 67.

23(4):263-271.

641, 1-11.

thresholds for male fertility potential of the other assays still need to be clarified.

stress should be performed.

Zool 284, 696-704.

Reprod 14, 2279-2285.

**13. References**

108.

also sperm DNA damage has been associated to recurrent pregnancy loss (Evenson et al., 1999; Carrell et al., 2003). In a population of first pregnancy planners Evenson et al. (1999) reported a miscarriage rate higher in fertile couples where the partner had a high SCSA-DFI compared to those with a low DFI. Also Carrell et al. (2003) by using the TUNEL assay on sperm from 24 couples with unexplained recurrent pregnancy loss showed that pregnancy loss was associated with DNA damage. As control group they used donors of known fertility and unscreened men from the general population.

Several studies have reported an increased risk of pregnancy loss after IVF and ICSI (Check et al., 2005; Zini et al. 2005; Lin et al., 2008), however data are conflicting (Bungum et al., 2007). In a recent meta-analysis Zini and co-workers (Zini et al., 2008) collected data from 11 studies involving 1549 IVF/ICSI treatments and 640 pregnancies. They demonstrated a combined odds ratio (OR) of 2.48 (95% CI; 1.52, 4.04, p<0.0001). The conclusions from this analysis should, however, be interpreted with care since different types of sperm DNA integrity tests and different sperm sources were included. We have demonstrated that washed semen is not predictive for the outcome of ART (Bungum et al., 2008). Further largescale studies should be performed to get firm conclusions.

#### **11. Prevention or treatment of sperm DNA damage**

A more precise diagnosing would enable clinicians to better counsel the infertile couple and may also result in improvement and further development of cause-related therapy, which is very little used in today's clinical practice (Skakkebaek et al., 1994). The effects of therapy based on antioxidants on sperm DNA quality has been evaluated, however, the studies have been small and conflicting (Greco et al., 2005a; Greco et al., 2005b; Silver et al., 2005; Song et al., 2006; Menezo et al., 2007; Kefer et al., 2009) and so far no standardized, clinically wellproven treatment of sperm oxidative stress and DNA damage are established.

Data on how to best advise couples in what they self can do to prevent against sperm DNA damage are lacking. Several life-style factors are suggested to negatively influence sperm DNA, however, data are conflicting. Examples are smoking and obesity (Reviewed in (DuPlessis et al., 2010 and Agarwal et al., 2008)). Interestingly, the first published intervention study in severe obese men demonstrated improvement in WHO parameters but not in sperm DNA integrity after weight reduction (Hakonsen et al., 2011).

#### **12. Conclusion and future perspectives**

Sperm DNA damage is a frequent problem in infertile men. Although not yet routine, sperm DNA integrity is a good tool in investigation and treatment of infertility. Sperm DNA integrity is a marker of male fertility, alone or in combination with the WHO semen parameters, this in natural conception as well as in ART.

The role of DFI as a predictor of fertilisation, embryo development and pregnancy in IVF and ICSI is still discussed. Although not conclusive, also a relation between sperm DNA damage and risk of pregnancy loss have been suggested.

Among the different sperm DNA integrity assays available SCSA is currently the only method that has provided clear and stable clinical cut-off levels and therefore can be recommended for a clinical sperm DNA damage evaluation. The normality ranges and thresholds for male fertility potential of the other assays still need to be clarified.

It seems clear that ART, especially ICSI, are able to overcome the natural barriers of sperm DNA damage levels not compatible with fertilisation under natural circumstances. The consequences of this for the progeny are still not clear, however, it is also reason to concern regarding possible consequences of achieving a pregnancy using spermatozoa possessing DNA damage. So far no proven treatment of sperm DNA damage is available. Adequately powered, placebo-controlled trials of antioxidants in the prevention of sperm oxidative stress should be performed.

#### **13. References**

30 Embryology – Updates and Highlights on Classic Topics

also sperm DNA damage has been associated to recurrent pregnancy loss (Evenson et al., 1999; Carrell et al., 2003). In a population of first pregnancy planners Evenson et al. (1999) reported a miscarriage rate higher in fertile couples where the partner had a high SCSA-DFI compared to those with a low DFI. Also Carrell et al. (2003) by using the TUNEL assay on sperm from 24 couples with unexplained recurrent pregnancy loss showed that pregnancy loss was associated with DNA damage. As control group they used donors of known

Several studies have reported an increased risk of pregnancy loss after IVF and ICSI (Check et al., 2005; Zini et al. 2005; Lin et al., 2008), however data are conflicting (Bungum et al., 2007). In a recent meta-analysis Zini and co-workers (Zini et al., 2008) collected data from 11 studies involving 1549 IVF/ICSI treatments and 640 pregnancies. They demonstrated a combined odds ratio (OR) of 2.48 (95% CI; 1.52, 4.04, p<0.0001). The conclusions from this analysis should, however, be interpreted with care since different types of sperm DNA integrity tests and different sperm sources were included. We have demonstrated that washed semen is not predictive for the outcome of ART (Bungum et al., 2008). Further large-

A more precise diagnosing would enable clinicians to better counsel the infertile couple and may also result in improvement and further development of cause-related therapy, which is very little used in today's clinical practice (Skakkebaek et al., 1994). The effects of therapy based on antioxidants on sperm DNA quality has been evaluated, however, the studies have been small and conflicting (Greco et al., 2005a; Greco et al., 2005b; Silver et al., 2005; Song et al., 2006; Menezo et al., 2007; Kefer et al., 2009) and so far no standardized, clinically well-

Data on how to best advise couples in what they self can do to prevent against sperm DNA damage are lacking. Several life-style factors are suggested to negatively influence sperm DNA, however, data are conflicting. Examples are smoking and obesity (Reviewed in (DuPlessis et al., 2010 and Agarwal et al., 2008)). Interestingly, the first published intervention study in severe obese men demonstrated improvement in WHO parameters

Sperm DNA damage is a frequent problem in infertile men. Although not yet routine, sperm DNA integrity is a good tool in investigation and treatment of infertility. Sperm DNA integrity is a marker of male fertility, alone or in combination with the WHO semen

The role of DFI as a predictor of fertilisation, embryo development and pregnancy in IVF and ICSI is still discussed. Although not conclusive, also a relation between sperm DNA

Among the different sperm DNA integrity assays available SCSA is currently the only method that has provided clear and stable clinical cut-off levels and therefore can be

proven treatment of sperm oxidative stress and DNA damage are established.

but not in sperm DNA integrity after weight reduction (Hakonsen et al., 2011).

fertility and unscreened men from the general population.

scale studies should be performed to get firm conclusions.

**11. Prevention or treatment of sperm DNA damage**

**12. Conclusion and future perspectives**

parameters, this in natural conception as well as in ART.

damage and risk of pregnancy loss have been suggested.

Role of Sperm DNA Integrity in Fertility 33

Duran, E. H., Morshedi, M., Taylor, S. and Oehninger, S. (2002) Sperm DNA quality predicts

Enciso, M., Muriel, L., Fernández, J.L., Goyanes, V., Segrelles, E., Marcos, M., Montejo, J.M.,

Erenpreiss, J., Bungum, M., Spano, M., Elzanaty, S., Orbidans, J. & Giwercman , A. (2006).

Erenpreiss, J., Jepson, K., Giwercman, A., Tsarev, I., Erenpreisa, J. & Spanò , M. (2004).

Erenpreiss, J., Spano, M., Erenpreisa, J., Bungum, M. & Giwercman, A. (2006a). Sperm

Evenson, D. & Jost, L. (2000). Sperm chromatin structure assay is useful for fertility

Evenson, D. & Wixon, R. (2006).Meta-analysis of sperm DNA fragmentation using the sperm chromatin structure assay. Reprod Biomed Online 12, 466-472. Evenson, D. P., Darzynkiewicz, Z. & Melamed, M. R. (1980). Relation of mammalian sperm

Evenson, D. P., Jost, L. K., Baer, R. K., Turner, T. W. &Schrader, S. M. (1991). Individuality of

Evenson, D. P., Jost, L. K., Marshall, D., Zinaman, M. J., Clegg, E., Purvis, K., de Angelis, P.

Evenson, D. P., Larson, K. L. & Jost, L. K. (2002).Sperm chromatin structure assay: its clinical

Fairbairn, D.W., Olive, P.L. & O'Neill K.L.. (1995). The COMET assay: a comprehensive

Fernández, J.L., Muriel, L., Goyanes, V., Segrelles, E., Gosálvez,J., Enciso, M., LaFromboise,

DNA denaturation patterns in human sperm as measured by the sperm chromatin

& Claussen, O. P. (1999). Utility of the sperm chromatin structure assay as a diagnostic and prognostic tool in the human fertility clinic. Hum Reprod 14, 1039-

use for detecting sperm DNA fragmentation in male infertility and comparisons

M. & De Jonge C. (2005). Simple determination of human sperm DNA fragmentation with an improved sperm chromatin dispersion test. Fertil

chromatin heterogeneity to fertility. Science 210, 1131-1133.

evidenced by the sperm chromatin dispersion test. J Androl 27:106-111. Erenpreisa, J., Erenpreiss, J., Freivalds, T., Slaidina, M., Krampe, R., Butikova, J., Ivanov, A.

of image cytometry algorithm. Cytometry A 52, 19-27.

infertile couples. Asian J Androl 10:786-790.

assessment. Methods Cell Sci 22, 169-189.

structure assay. Reprod Toxicol 5, 115-125.

with other techniques. J Androl 23, 25-43.

review. Mutat Res 339(1):37-59.

Steril;84:833-842.

3122-3128.

19:2277–2282.

Androl 8, 11-29.

1049.

intrauterine insemination outcome: a prospective cohort study. Hum Reprod 17,

Ardoy, M., Pacheco, A. & Gosálvez J. (2006). Infertile men with varicocele show a high relative proportion of sperm cells with intense nuclear damage level,

& Pjanova, D. (2003). Toluidine blue test for sperm DNA integrity and elaboration

Intra-individual variation in Sperm Chromatin Structure Assay parameters in men from infertile couples: clinical implications. Human Reproduction 21:2061-2064. Erenpreiss, J., Elzanaty, S. & Giwercman, A. (2008). Sperm DNA damage in men from

Toluidine blue cytometry test for sperm DNA conformation: comparison with the flow cytometric sperm chromatin structure and TUNEL assays. Hum Reprod

chromatin structure and male fertility: biological and clinical aspects. Asian J

Boe-Hansen, G. B., Fedder, J., Ersboll, A. K. & Christensen, P. (2006) The sperm chromatin

Bonde, J. P., Ernst, E., Jensen, T. K., Hjollund, N. H., Kolstad, H., Henriksen, T. B., Scheike,

Bungum, M., Humaidan, P., Axmon, A., Spanó, M., Bungum, L., Erenpreiss, J. & Giwercman

Bungum, M., Humaidan, P., Spanó, M., Jepson, K., Bungum, L. & Giwercman, A. (2004).The

Carrell, D. T. & Liu, L. (2001) Altered protamine 2 expression is uncommon in donors of

Carrell, D. T., Liu, L., Peterson, C. M., Jones, K. P., Hatasaka, H. H., Erickson, L. & Campbell,

Check, J. H., Graziano, V., Cohen, R., Krotec, J. & Check, M. L. (2005). Effect of an abnormal

Chen, Z., Toth, T., Godfrey-Bailey, L., Mercedat, N., Schiff, I. & Hauser, R. (2003) Seasonal

Chia, S. E., Tay, S. K.& Lim, S. T. (1998) What constitutes a normal seminal analysis? Semen

Collins, A.R. (2004). The COMET assay for DNA damage and repair: principles,

Collins, J. A., Barnhart, K. T. & Schlegel, P. N. (2008). Do sperm DNA integrity tests predict

Cooper, T. G., Neuwinger, J., Bahrs, S. & Nieschlag, E. (1992). Internal quality control of

Donnelly, E. T., O'Connell, M., McClure, N. & Lewis, S. E. (2000). Differences in nuclear

Du Plessis SS, Cabler S, McAlister DA, Sabanegh E, Agarwal A. (2010). The effect of obesity

on sperm disorders and male infertility. Nat Rev Urol. 7(3):153-161

DNA fragmentation and mitochondrial integrity of semen and prepared human

reflect other abnormalities of spermiogenesis. J Androl 22, 604-610.

with ICSI in previous IVF failures. Arch Androl 51, 121-124.

parameters of 243 fertile men. Hum Reprod 13, 3394-3398.

applications, and limitations. Mol Biotechnol 26(3):249-261.

pregnancy with in vitro fertilization? Fertil Steril 89, 823-831.

semen analysis. Fertil Steril 58, 172-178.

spermatozoa. Hum Reprod 15:1552–1561.

recurrent pregnancy loss. Arch Androl 49, 49-55.

technology outcome. Human Reproduction 22:174-179.

1576-1582.

23:4-10.

231.

Lancet 352, 1172-1177.

structure assay as a diagnostic tool in the human fertility clinic. Hum Reprod 21,

T., Giwercman, A., Olsen, J. & Skakkebaek, N. E. (1998). Relation between semen quality and fertility: a population-based study of 430 first-pregnancy planners.

A. (2007) Sperm DNA integrity assessment in prediction of assisted reproduction

predictive value of sperm chromatin structure assay (SCSA) parameters for the outcome of intrauterine insemination, IVF and ICSI. Hum Reprod 19:1401-1408. Bungum, M., Spanó, M., Humaidan, P., Eleuteri, P., Rescia, M. & Giwercman, A. (2008).

Sperm Chromatin Structure Assay (SCSA) parameters measured after density gradient centrifugation are not predictive for the outcome of ART. Hum Reprod

known fertility, but common among men with poor fertilizing capacity, and may

B. (2003). Sperm DNA fragmentation is increased in couples with unexplained

sperm chromatin structural assay (SCSA) on pregnancy outcome following (IVF)

variation and age-related changes in human semen parameters. J Androl 24, 226-

Role of Sperm DNA Integrity in Fertility 35

Hammadeh, M. E., Zeginiadov, T., Rosenbaum, P., Georg, T., Schmidt, W. & Strehler, E.

Hammadeh, M.E., Stieber, M., Haidl, G. & Schmidt W. (1998). Association between sperm

cleavage and pregnancy rates in an IVF program. Andrologia 30(1):29-35. Henkel, R., Hajimohammad, M., Stalf, T., Hoogendijk, C., Mehnert, C., Menkveld, R., Gips,

Hewitson, L., Simerly, C, Sutovsky, P., Dominko, T., Takahashi, D. & Schatten, G. (1999).

Host, E., Lindenberg, S. & Smidt-Jensen, S. (2000). The role of DNA strand breaks in human spermatozoa used for IVF and ICSI. Acta Obstet Gynecol Scand 79(7):559-563. Huang, C. C., Lin, D. P., Tsao, H. M., Cheng, T. C., Liu, C. H. & Lee, M. S. (2005). Sperm

Huszar, G., Zeyneloglu, H. B. & Vigue, L. (1999). Cellular maturity and fertilizing potential

Irvine, D. S., Twigg, J. P., Gordon, E. L., Fulton, N., Milne, P. A. & Aitken, R. J. (2000). DNA

Jorgensen, N., Andersen, A. G., Eustache, F., Irvine, D. S., Suominen, J., Petersen, J. H.,

Jorgensen, N., Asklund, C., Carlsen, E. & Skakkebaek, N. E. (2006). Coordinated European

Keel, B. A. (2006). Within- and between-subject variation in semen parameters in infertile

Kefer, J.C., Agarwal, A., & Sabanegh, E. (2009), Role of antioxidants in the treatment of male

Kurinczuk, J. J. (2003) Safety issues in assisted reproduction technology. From theory to

Larson, K. L., DeJonge, C. J., Barnes, A. M., Jost, L. K.& Evenson, D. P. (2000). Sperm

differences in semen quality in Europe. Hum Reprod 16, 1012-1019.

a matter of concern. Int J Androl 29, 54-61; discussion 105-108.

fertility and should we be concerned? Hum Reprod 18, 925-931.

assisted reproductive techniques. Hum Reprod 15, 1717-1722.

men and normal semen donors. Fertil Steril 85, 128-134.

infertility. Int J Urol 2009;16:449-457.

a cohort of severely obese men. Reprod Health 17;8:24.

the assessment of male fertility. Arch Androl 46, 99-104.

on fertilization and pregnancy. Fertil Steril 81, 965-972.

might not affect pregnancy rates. Fertil Steril 84, 130-140.

Cache River Press, Vienna.

385–396.

44.

Does weight loss improve semen quality and reproductive hormones? results from

(2001). Predictive value of sperm chromatin condensation (aniline blue staining) in

cell chromatin condensation, morphology based on strict criteria, and fertilization,

H., Schill, W. B. & Kruger, T. F. (2004). Influence of deoxyribonucleic acid damage

The fate of sperm components within the egg during fertilization: implications for infertility. In C, G. (ed) *The Male Gamete: From Basic Science to Clinical Applications*

DNA fragmentation negatively correlates with velocity and fertilization rates but

of sperm populations in natural and assisted reproduction. In C, G. (ed) *The Male Gamete: From Basic Science to Clinical Applications*. Cache River Press, Vienna, pp.

integrity in human spermatozoa: relationships with semen quality. J Androl 21, 33-

Andersen, A. N., Auger, J., Cawood, E. H. & Horte, A. *et al.* (2001). Regional

investigations of semen quality: results from studies of Scandinavian young men is

reality--just what are the data telling us about ICSI offspring health and future

chromatin structure assay parameters as predictors of failed pregnancy following

Fernández, J.L., Muriel, L., Rivero, M.T., Goyanes, V., Vazquez, R., Alvarez, J.G. (2003). The

Fuentes-Mascorro, G., Vergara-Onofre, M., Mercado, E., Hernandez-Perez, O.& Rosado, A.

Gandini, L., Lombardo, F., Paoli, D., Caponecchia, L., Familiari, G., Verlengia, C., Dondero,

Gandini, L., Lombardo, F., Paoli, D., Caruso, F., Eleuteri, P., Leter, G., Ciriminna, R.,

Giwercman, A., Lindstedt, L., Larsson, M., Bungum, M., Spanó, M., Levine, R.J. & Rylander,

Giwercman, A., Richthoff, J., Hjollund, H., Bonde, J. P., Jepson, K., Frohm, B. & Spano, M.

Giwercman, A., Spano, M., Lahdetie, J. & Bonde, J. P. (1999). Quality assurance of semen

Gorczyca, W., Gong, J. & Darzynkiewicz, Z. (1993). Detection of DNA strand breaks in

Govaerts, I., Koenig, I., Van den Bergh, M., Bertrand, E., Revelard, P. & Englert, Y. (1996) Is

Greco, E., Iacobelli, M., Rienzi, L., Ubaldi, F., Ferrero, S. & Tesarik, J. (2005). Reduction of

Greco, E., Romano, S., Iacobelli, M-, Ferrero, S., Baroni, E., Minasi, M..G., Ubaldi, F., Rienzi

Griffin, D. K., Hyland, P., Tempest, H. G. & Homa, S. T. (2003). Safety issues in assisted

Håkonsen, L.B., Thulstrup, A.M., Aggerholm, A.S., Olsen, J., Bonde, J.P., Andersen, C.Y.,

chromosome abnormalities in their sperm? Hum Reprod 18, 229-235. Guzick, D. S., Overstreet, J. W., Factor-Litvak, P., Brazil, C. K., Nakajima, S. T., Coutifaris, C.,

DNA fragmentation. J Androl 24:59–66.

spermatozoa. Hum Reprod 15, 830-839.

parameters. Fertil Steril 80, 1404-1412.

23-25; discussion 76-28.

26, 349-353.

345, 1388-1393.

in vivo: a case-control study. Int J Androl 33:221-227.

nick translation assays. Cancer Res 53, 1945-1951.

antioxidant treatment. Hum Reprod 20:2590-2594.

early pregnancy data about ICSI? Hum Reprod 11, 440-443.

Androl 45, 61-71.

1417.

sperm chromatin dispersion test: a simple method for the determination of sperm

(2000). Participation of DNA structure on sperm chromatin organization. Arch

F. & Lenzi, A. (2000). Study of apoptotic DNA fragmentation in human

Culasso, F., Dondero, F.& Lenzi, A. *et al.* (2004). Full-term pregnancies achieved with ICSI despite high levels of sperm chromatin damage. Hum Reprod 19, 1409-

L. (2010). Sperm chromatin structure assay as an independent predictor of fertility

(2003). Correlation between sperm motility and sperm chromatin structure assay

analysis in multicenter studies. Asclepios. Scand J Work Environ Health 25 Suppl 1,

individual apoptotic cells by the in situ terminal deoxynucleotidyl transferase and

intracytoplasmic sperm injection (ICSI) a safe procedure? What do we learn from

the incidence of sperm DNA fragmentation by oral antioxidant treatment. J Androl

L. & Tesarik J. (2005). ICSI in cases of sperm DNA damage: beneficial effect of oral

reproduction technology: Should men undergoing ICSI be screened for

Carson, S. A., Cisneros, P., Steinkampf, M. P. & Hill, J. A. *et al.* (2001). Sperm morphology, motility, and concentration in fertile and infertile men. N Engl J Med

Bungum, M., Ernst, E.H., Hansen, M.L., Ernst, E.H. & Ramlau-Hansen CH (2011).

Does weight loss improve semen quality and reproductive hormones? results from a cohort of severely obese men. Reprod Health 17;8:24.

Role of Sperm DNA Integrity in Fertility 37

Muratori, M., Maggi, M., Spinelli, S., Filimberti, E., Forti, G. and Baldi, E. (2003)

Nallella, K. P., Sharma, R. K., Aziz, N. and Agarwal, A. (2006) Significance of sperm characteristics in the evaluation of male infertility. Fertil Steril 85, 629-634. Nasr-Esfahani, M. H., Salehi, M., Razavi, S., Anjomshoa, M., Rozbahani, S., Moulavi, F. &

Neuwinger, J., Behre, H. M. and Nieschlag, E. (1990) External quality control in the andrology laboratory: an experimental multicenter trial. Fertil Steril 54, 308-314. O'Flynn O'Brien KL, Varghese AC, Agarwal A. The genetic cause of male factor infertility: a

Olechuk, K., Giwercman, A & Bungum M. (2011). Intra-individual variation of the sperm

Payne, J. F., Raburn, D. J., Couchman, G. M., Price, T. M., Jamison, M. G. and Walmer, D. K.

Perera,. D, Pizzey, A., Campbell, A., Katz, M., Porter, J., Petrou, M., Irvine, D.S. & Chatterjee

Potts, R. J., Newbury, C. J., Smith, G., Notarianni, L. J. & Jefferies, T. M. (1999). Sperm chromatin damage associated with male smoking. Mutat Res 423, 103-111.

Richthoff, J., Spano, M., Giwercman, Y. L., Frohm, B., Jepson, K., Malm, J., Elzanaty, S.,

Robbins, W. A., Vine, M. F., Truong, K. Y. & Everson, R. B. (1997). Use of fluorescence in situ

Sakkas, D. & Alvarez J.G. (2010). Sperm DNA fragmentation: mechanisms of origin, impact

Sakkas, D. & Alvarez, J.G. (2010). Sperm DNA fragmentation: mechanisms of origin, impact

Sakkas, D., Mariethoz, E., Manicardi, G., Bizzaro, D., Bianchi, P. G. & Bianchi, U. (1999). Origin of DNA damage in ejaculated human spermatozoa. Rev Reprod 4, 31-37.

on reproductive outcome, and analysis. Fertil Steril 93:1027-1036.

on reproductive outcome, and analysis. Fertil Steril 93:1027-1036.

of assisted reproductive techniques. Fertil Steril 84, 356-364.

patients with iron overload. Hum Reprod 17:1820-1825.

Rai, R. & Regan, L. (2006). Recurrent miscarriage. Lancet 368, 601-611

structure assay (SCSA). Hum Reprod 17, 3162-3169.

disomy in teenage men. Fertil Steril 70, 715-723.

long term incubation. J Androl 24, 253-262.

review. Fertil Steril 2010;93:1–12.

spermatozoon into an oocyte. Lancet 340, 17 .

205.

Spontaneous DNA fragmentation in swim-up selected human spermatozoa during

Mardani, M. (2005). Effect of sperm DNA damage and sperm protamine deficiency on fertilization and embryo development post-ICSI. Reprod Biomed Online 11, 198-

chromatin structure assay DNA fragmentation index in men from infertile couples. Hum. Reprod. doi: 10.1093/humrep/der328Palermo, G., Joris, H., Devroey, P. and Van Steirteghem, A. C. (1992) Pregnancies after intracytoplasmic injection of single

(2005) Redefining the relationship between sperm deoxyribonucleic acid fragmentation as measured by the sperm chromatin structure assay and outcomes

R. (2002). Sperm DNA damage in potentially fertile homozygous beta-thalassaemia

Stridsberg, M. and Giwercman, A. (2002) The impact of testicular and accessory sex gland function on sperm chromatin integrity as assessed by the sperm chromatin

hybridization (FISH) to assess effects of smoking, caffeine, and alcohol on aneuploidy load in sperm of healthy men. Environ Mol Mutagen 30, 175-183. Rubes, J., Lowe, X., Moore, D., 2nd, Perreault, S., Slott, V., Evenson, D., Selevan, S. G. &

Wyrobek, A. J. (1998). Smoking cigarettes is associated with increased sperm

Larson-Cook, K. L., Brannian, J. D., Hansen, K. A., Kasperson, K. M., Aamold, E. T. &

Lewis, S. E & Agbaje, I. M. (2008). Using the alkaline comet assay in prognostic tests for

Lewis, S. E. (2007). Is sperm evaluation useful in predicting human fertility? Reproduction

Lewis, S. E.& Aitken, R. J. (2005) DNA damage to spermatozoa has impacts on fertilization

Lewis, S. E., O'Connell, M., Stevenson, M., Thompson-Cree, L. & McClure, N. (2004). An

Li, Z., Wang, L., Cai, J. & Huang, H. (2006). Correlation of sperm DNA damage with IVF

Lin, M. H., Kuo-Kuang Lee, R., Li, S. H., Lu, C. H., Sun, F. J. and Hwu, Y. M. (2007). Sperm

Lopes, S., Sun, J. G., Jurisicova, A., Meriano, J. & Casper, R. F. (1998). Sperm

Makhlouf, A. A. and Niederberger, C. (2006) DNA integrity tests in clinical practice: it is not a simple matter of black and white (or red and green). J Androl 27, 316-323. Marchetti, F. and Wyrobek, A. J. (2005) Mechanisms and consequences of paternally-

Matsuda, Y. and Tobari, I. (1988) Chromosomal analysis in mouse eggs fertilized in vitro

Matzuk,. MM. & Lamb, D.J. (2008), The biology of infertility: research advances and clinical

Ménézo, Y.J., Hazout, A., Panteix , G., Robert, F., Rollet, J., Cohen-Bacrie, P., Chapuis, F.,

methanesulfonate (MMS and EMS). Mutat Res 198, 131-144.

challenges. Nat Med 14:1197–1213.

structure assay. Fertil Steril 80, 895-902.

and pregnancy. Cell Tissue Res 322, 33-41.

23:163-170.

134, 31-40.

1394.

359

23, 367-376.

Steril 69, 528-532.

112-129.

Evenson, D. P. (2003). Relationship between the outcomes of assisted reproductive techniques and sperm DNA fragmentation as measured by the sperm chromatin

male infertility and assisted reproductive technology outcomes. Mutagenesis

algorithm to predict pregnancy in assisted reproduction. Hum Reprod 19, 1385-

and ICSI outcomes: a systematic review and meta-analysis. J Assist Reprod Genet

chromatin structure assay parameters are not related to fertilization rates, embryo quality, and pregnancy rates in in vitro fertilization and intracytoplasmic sperm injection, but might be related to spontaneous abortion rates. Fertil Steril 90(2):352-

deoxyribonucleic acid fragmentation is increased in poor-quality semen samples and correlates with failed fertilization in intracytoplasmic sperm injection. Fertil

transmitted chromosomal abnormalities. Birth Defects Res C Embryo Today 75,

with sperm exposed to ultraviolet light (UV) and methyl and ethyl

Clément,. P. & Benkhalifa, M. (2007). Antioxidants to reduce sperm DNA fragmentation: an unexpected adverse effect. Reprod Biomed Online 14:418-421. Morris, I. D., Ilott, S., Dixon, L. & Brison, D. R. (2002). The spectrum of DNA damage in

human sperm assessed by single cell gel electrophoresis (Comet assay) and its relationship to fertilization and embryo development. Hum Reprod 17, 990-998.

Role of Sperm DNA Integrity in Fertility 39

Spano, M., Bonde, J. P., Hjollund, H. I., Kolstad, H. A., Cordelli, E. and Leter, G. (2000)

Spano, M., Kolstad, A. H., Larsen, S. B., Cordelli, E., Leter, G., Giwercman, A. and Bonde, J.

Sun, J. G., Jurisicova, A. & Casper, R. F. (1997). Detection of deoxyribonucleic acid

Swan, S. H. (2006) Semen quality in fertile US men in relation to geographical area and

The Practice Committee of the American Society of Reproductive Medicine (2008). The clinical utility of sperm DNA integrity testing. Fertil Steril 90 (Suppl 5):178-180. Tomlinson, M. J., Moffatt, O., Manicardi, G. C., Bizzaro, D., Afnan, M. & Sakkas, D. (2001).

Tomsu, M., Sharma, V. & Miller, D. (2002). Embryo quality and IVF treatment outcomes

Varghese, A. C., Goldberg, E. and Agarwal, A. (2007) Current and future perspectives on

Ward, W. S. (1993). Deoxyribonucleic acid loop domain tertiary structure in mammalian

Ward, W. S. and Coffey, D. S. (1991). DNA packaging and organization in mammalian spermatozoa: comparison with somatic cells. Biol Reprod 44, 569-574. WHO (2000). WHO Manual for the Standardised Investigation, Diagnosis and Management

Wolf, J.P., Bulwa, S., Ducot, B., Rodrigues, D. & Jouannet, P. (1996). Fertilizing ability of

World Health Organization. WHO laboratory manual for the examination of human semen

World Health Organization. WHO laboratory manual for the examination and processing of

sperm with unexplained in vitro fertilization failures, as assessed by the zona-free hamster egg penetration assay: its prognostic value for sperm-oolemma interaction.

and sperm-cervical mucus interaction. Cambridge: Cambridge University Press;

of the Infertile Male. Cambridge University Press, Cambridge.

human semen. 5 ed: World Health Organization; 2010.

Verpoest, W. & Tournaye, H. (2006). ICSI: hype or hazard? Hum Fertil (Camb) 9, 81-92. Virro, M. R., Larson-Cook, K. L. & Evenson, D. P. (2004). Sperm chromatin structure assay

in epidemiological studies. Asclepios. Hum Reprod 13, 2495-2505..

pesticide exposure. Int J Androl 29, 62-68; discussion 105-108.

Planner Study Team. Fertil Steril 73, 43-50.

conception. Hum Reprod 16, 2160-2165.

spermatozoa. Biol Reprod 48, 1193-1201.

56, 602-607.

1862.

719-727.

1999.

Fertil Steril 81, 1289-1295.

Fertil Steril 65(6):1196-1201.

Sperm chromatin damage impairs human fertility. The Danish First Pregnancy

P. (1998) The applicability of the flow cytometric sperm chromatin structure assay

fragmentation in human sperm: correlation with fertilization in vitro. Biol Reprod

Interrelationships between seminal parameters and sperm nuclear DNA damage before and after density gradient centrifugation: implications for assisted

may correlate with different sperm comet assay parameters. Hum Reprod 17, 1856-

intracytoplasmic sperm injection: a critical commentary. Reprod Biomed Online 15,

(SCSA) parameters are related to fertilization, blastocyst development, and ongoing pregnancy in in vitro fertilization and intracytoplasmic sperm injection cycles.

Sakkas, D., Moffatt, O., Manicardi, G. C., Mariethoz, E., Tarozzi, N. and Bizzaro, D. (2002)

Sakkas, D., Urner, F., Bianchi, P. G., Bizzaro, D., Wagner, I., Jaquenoud, N., Manicardi, G. &

Saleh, R. A., Agarwal, A., Nada, E. A., El-Tonsy, M. H., Sharma, R. K., Meyer, A., Nelson, D.

Saleh, R. A., Agarwal, A., Nelson, D. R., Nada, E. A., El-Tonsy, M. H., Alvarez, J. G.,

Sega, G. A., Sotomayor, R. E. & Owens, J. G. (1978). A study of unscheduled DNA synthesis induced by X-rays in the germ cells of male mice. Mutat Res 49, 239-257. Selevan, S. G., Borkovec, L., Slott, V. L., Zudova, Z., Rubes, J., Evenson, D. P. & Perreault, S.

Seli, E., Gardner, D.K., Schoolcraft, W.B, Moffatt, O. & Sakkas, D. (2004). Extent of nuclear

Silver, E. W., Eskenazi, B., Evenson, D. P., Block, G., Young, S. & Wyrobek, A. J. (2005).

Simon, L., Brunborg, G., Stevenson, M., Lutton, D., McManus, J. & Lewis, S.E. (2010).

Simon, L., Lutton, D., McManus, J. & Lewis SE. (2011). Sperm DNA damage measured by

Singh, N. P., Muller, C. H. and Berger, R. E. (2003) Effects of age on DNA double-strand

Skakkebaek, N. E., Giwercman, A. and de Kretser, D. (1994) Pathogenesis and management

Smit, M., Dohle, G. R., Hop, W. C., Wildhagen, M. F., Weber, R. F. and Romijn, J. C. (2007)

breaks and apoptosis in human sperm. Fertil Steril 80, 1420-1430.

sperm DNA integrity in infertile men. Int J Androl. 2006;29:569-75.

seasonal air pollution. Environ Health Perspect 108, 887-894.

after intracytoplasmic sperm injection. Hum Reprod 11, 837-843.

involvement of apoptosis. Biol Reprod 66, 1061-1067.

infertility. Fertil Steril 79 Suppl 3, 1597-1605.

in vitro fertilization. Fertil Steril 82:378-383.

vitro fertilization success. Fertil Steril 95(2):652-657.

of male infertility. Lancet 343, 1473-1479.

Pharmacol 196, 108-113.

men. J Androl 26, 550-556.

Hum Reprod 25(7):1594-1608.

Nature of DNA damage in ejaculated human spermatozoa and the possible

Campana, A. (1996). Sperm chromatin anomalies can influence decondensation

R. and Thomas, A. J. (2003a) Negative effects of increased sperm DNA damage in relation to seminal oxidative stress in men with idiopathic and male factor

Thomas, A. J., Jr. and Sharma, R. K. (2002b) Increased sperm nuclear DNA damage in normozoospermic infertile men: a prospective study. Fertil Steril 78, 313-318. Sanchez-Pena, L. C., Reyes, B. E., Lopez-Carrillo, L., Recio, R., Moran-Martinez, J., Cebrian,

M. E. & Quintanilla-Vega, B. (2004). Organophosphorous pesticide exposure alters sperm chromatin structure in Mexican agricultural workers. Toxicol Appl

D. (2000). Semen quality and reproductive health of young Czech men exposed to

DNA damage in ejaculated spermatozoa impacts on blastocyst development after

Effect of antioxidant intake on sperm chromatin stability in healthy nonsmoking

Clinical significance of sperm DNA damage in assisted reproduction outcome.

the alkaline Comet assay as an independent predictor of male infertility and in

Clinical correlates of the biological variation of sperm DNA fragmentation in infertile men attending an andrology outpatient clinic. Int J Androl 30, 48-55. Song, G.J., Norkus, E.P.& Lewis, V. (2006). Relationship between seminal ascorbic acid and

**3**

*Brazil*

**The Epididymis:**

**Embryology, Structure, Function and**

**Its Role in Fertilization and Infertility**

Marilia Lopes Justino and Luís Antonio Violin Pereira

*State University of Campinas (UNICAMP)*

Kélen Fabiola Arrotéia, Patrick Vianna Garcia, Mainara Ferreira Barbieri,

The epididymis, located between the efferent ducts and the vas deferens, is a male accessory organ characterized by a single coiled tubule duct with an estimated length of 5–7 m in men

The anatomic segments of the epididymis include the initial segment, the caput, the corpus and the cauda. Each region consists of a lumen and a polarized epithelium composed mostly of principal and basal cells (Lasserre et al., 2001; Dacheux et al., 2005). Although these four anatomical regions of the epididymis are easily identified in most adult male mammals (Yanagimachi et al., 1985; Smithwick & Young, 2001), histological and ultrastructural segmentation of this organ varies among the different phylogenies of mammals. The rat epididymis is most commonly adopted as an experimental model of study (Figure 1).

Several descriptive anatomical and histological studies of the epididymis appeared at the beginning of the twentieth century. The authors hypothesized that the epididymal secretions played a role in the maintenance of sperm vitality, sperm motility (Benoit, 1926) and the capacity to become fertile (Young, 1929a, 1929b, 1931). Relatively little research was done on the excurrent duct system during the ensuing three decades. However, in 1967, Marie-Claire Orgebin-Crist demonstrated that the key event in sperm maturation was not the passage of time but the exposure of the sperm to the luminal environment of the

The epididymal duct is now recognized as a channel that transports, concentrates and stores the spermatozoa. It is also known that the spermatozoa leaving the testis are immovable, immature and unable to fertilize an oocyte (Yanagimachi, 1994; Flesch & Gadella, 2000), and that under androgen control, the epididymal epithelium secretes proteins within the intraluminal compartment that create a very complex environment surrounding the spermatozoa (Hermo et al., 1994, 2004; Sullivan, 2004). This luminal compartment stores the spermatozoa until ejaculation and specifically prepares the sperm for fertilization by providing the essentials in terms of temperature, oxygen tension, pH and an available energy substrate (Dacheux et al., 2005). The epididymal duct produces the morphological,

**1. Introduction**

(Sullivan, 2004; O'Hara et al., 2011).

epididymis (Bedford, 1967; Orgebin-Crist, 1967).

### **The Epididymis: Embryology, Structure, Function and Its Role in Fertilization and Infertility**

Kélen Fabiola Arrotéia, Patrick Vianna Garcia, Mainara Ferreira Barbieri, Marilia Lopes Justino and Luís Antonio Violin Pereira *State University of Campinas (UNICAMP) Brazil*

#### **1. Introduction**

40 Embryology – Updates and Highlights on Classic Topics

Zini A, Sigman M. Are tests of sperm DNA damage clinically useful? Pros and cons. J

Zini, A., Bielecki, R., Phang, D. and Zenzes, M. T. (2001a) Correlations between two markers

Zini, A., Boman, J.M., Belzile, E. & Ciampi, A. (2008). Sperm DNA damage is asociated with

Zini, A., Fischer, M. A., Sharir, S., Shayegan, B., Phang, D. and Jarvi, K. (2002). Prevalence of

Zini, A., Kamal, K., Phang, D., Willis, J. and Jarvi, K. (2001b) Biologic variability of sperm

DNA denaturation in infertile men. Urology 58, 258-261.

of sperm DNA integrity, DNA denaturation and DNA fragmentation, in fertile and

an increased risk of pregnancy loss after IVF and ICSI: systematic review and meta-

abnormal sperm DNA denaturation in fertile and infertile men. Urology 60, 1069-

Androl 2009;30:219-229.

1072.

infertile men. Fertil Steril 75, 674-677.

analysis. Hum Reprod 23 (1),4-10.

The epididymis, located between the efferent ducts and the vas deferens, is a male accessory organ characterized by a single coiled tubule duct with an estimated length of 5–7 m in men (Sullivan, 2004; O'Hara et al., 2011).

The anatomic segments of the epididymis include the initial segment, the caput, the corpus and the cauda. Each region consists of a lumen and a polarized epithelium composed mostly of principal and basal cells (Lasserre et al., 2001; Dacheux et al., 2005). Although these four anatomical regions of the epididymis are easily identified in most adult male mammals (Yanagimachi et al., 1985; Smithwick & Young, 2001), histological and ultrastructural segmentation of this organ varies among the different phylogenies of mammals. The rat epididymis is most commonly adopted as an experimental model of study (Figure 1).

Several descriptive anatomical and histological studies of the epididymis appeared at the beginning of the twentieth century. The authors hypothesized that the epididymal secretions played a role in the maintenance of sperm vitality, sperm motility (Benoit, 1926) and the capacity to become fertile (Young, 1929a, 1929b, 1931). Relatively little research was done on the excurrent duct system during the ensuing three decades. However, in 1967, Marie-Claire Orgebin-Crist demonstrated that the key event in sperm maturation was not the passage of time but the exposure of the sperm to the luminal environment of the epididymis (Bedford, 1967; Orgebin-Crist, 1967).

The epididymal duct is now recognized as a channel that transports, concentrates and stores the spermatozoa. It is also known that the spermatozoa leaving the testis are immovable, immature and unable to fertilize an oocyte (Yanagimachi, 1994; Flesch & Gadella, 2000), and that under androgen control, the epididymal epithelium secretes proteins within the intraluminal compartment that create a very complex environment surrounding the spermatozoa (Hermo et al., 1994, 2004; Sullivan, 2004). This luminal compartment stores the spermatozoa until ejaculation and specifically prepares the sperm for fertilization by providing the essentials in terms of temperature, oxygen tension, pH and an available energy substrate (Dacheux et al., 2005). The epididymal duct produces the morphological,

The Epididymis: Embryology, Structure, Function and Its Role in Fertilization and Infertility 43

between the epithelial cells and the contents of the luminal compartment remains challenging but important.  Additionally, up to 40% of infertile men exhibit idiopathic infertility that may reflect sperm maturational disorders

A greater understanding of the function and biochemical properties of the epididymis may lead to the development of therapeutic agents to treat certain types of infertility. This chapter provides an overview of the human epididymal embryology, malformation,

In mammals, the genetic sex of the embryo is determined mainly by the presence or absence of a single gene on the Y chromosome, the SRY, which is required to initiate male-specific pathways and to repress female-specific pathways of development (Moore & Persaud, 2003).  The gonads (testicles or ovaries), the genital ducts (Wolffian or Müllerian) and the urogenital sinus of both sexes emerge from the morphologically undifferentiated primordia. We can then assume that the genes involved in the establishment of these primordia are the same for both sexes. During development, the first morphological sex difference appear in the gonads of male embryos, followed by the genital ducts, and finally, they appear in the urogenital sinus (Larios & Mendoza, 2001).

In humans, the genital ducts arise from the intermediate mesoderm. Shortly after the formation of the Wolffian ducts, the adjacent Müllerian ducts are formed. In males, sexual differentiation develops through regression of the Müllerian ducts and the development of the Wolffian ducts into multiple reproductive organs, such as the epididymis, the vas deferens ducts and the the seminal vesicles. In females, the Wolffian ducts regress and the Müllerian ducts differentiate to form the uterus, the fallopian tubes, and the proximal

The development of sexual differentiation involves genetic processes that are primarily

In the male tract, Müllerian duct regression, which depends on the testicular Sertoli cells, is mediated by the Y chromosome genes and is triggered by a mechanism that is not well understood. The Sertoli cells produce and secrete an anti-Müllerian substance, a nonsteroidal hormone. Under the control of human chorionic gonadotrophin (hCG), a placental gonadotrophin, the Leydig cells then differentiate and produce the androgens that will positively regulate the ipsilateral Wolffian duct in a paracrine way. These hormonal combinations induce the cranial Wolffian duct to become highly convoluted and to

The major morphogenic event during Wolffian duct/epididymal duct development is the elongation and coiling of the duct (Hannema & Hughes, 2007). The process of elongation is likely a product of potential mechanisms such as cell proliferation coupled with directed cell rearrangements, along with the interactions between the Wolffian duct ephitelium and the

(Cornwall, 2009).

structure and function.

**2. The prenatal development of the epididymis**

vagina (Hannema & Hughes, 2007; Kobayashi & Behringer, 2003).

controlled by the sexual chromosomes (França et al., 2005).

surrounding mesenchyme cells (Hinton et al., 2011).

differentiate into the epididymis.

biochemical, physiological and functional changes to the structures of the spermatozoa through a process known as epididymal maturation, which converts the spermatozoa into fertilization-competent cells (Toshimori, 2003; Gatti et al., 2004; Flesch & Gadella, 2000).

Fig. 1. The regionalization of the epididymis. The illustrations compare the mouse and human epididymides. A) Mouse epididymis (A1: proximal caput, A2: mid-caput). B) Human epididymis (A1: anterior caput, A2: posterior caput, B1: anterior corpus, B2: midcorpus, B3: posterior corpus, C1: anterior cauda, C2: posterior cauda).

Since 1966, more than 12,000 research articles have been published on the epididymis. These articles, while addressing the various aspects of the epididymis from several points of view, all agree that the epididymis is crucial for the preparation of the spermatozoa prior to fertilization. It is well known that the proteins and small molecules secreted by the epididymal epithelium into the lumen interact with the transiting spermatozoa and directly or indirectly affect the spermatozoa surface; these proteins and small molecules function as signaling molecules to induce activity in the other epididymal proteins (Gatti et al., 2004). A significant number of these molecules will be taken up by the spermatozoa. Several of the acrosome molecules, which were previously formed during spermiogenesis and are involved in the acrosome reaction or in the sperm-zona pellucida interaction and sperm-egg fusion, will be gradually rearranged and compartmentalized in a stage-specific manner during sperm maturation (França et al., 2005).

Through research, we have learned a substantial amount about the function and the biochemical properties of the epididymal structure. However, there are still gaps in nearly all aspects of the research. Studying the complexity of the cellular properties, the spatial and temporal organization of protein syntheses and secretion and the dynamic interactions between the epithelial cells and the contents of the luminal compartment remains challenging but important.  Additionally, up to 40% of infertile men exhibit idiopathic infertility that may reflect sperm maturational disorders (Cornwall, 2009).

A greater understanding of the function and biochemical properties of the epididymis may lead to the development of therapeutic agents to treat certain types of infertility. This chapter provides an overview of the human epididymal embryology, malformation, structure and function.

#### **2. The prenatal development of the epididymis**

42 Embryology – Updates and Highlights on Classic Topics

biochemical, physiological and functional changes to the structures of the spermatozoa through a process known as epididymal maturation, which converts the spermatozoa into fertilization-competent cells (Toshimori, 2003; Gatti et al., 2004; Flesch & Gadella, 2000).

Fig. 1. The regionalization of the epididymis. The illustrations compare the mouse and human epididymides. A) Mouse epididymis (A1: proximal caput, A2: mid-caput). B) Human epididymis (A1: anterior caput, A2: posterior caput, B1: anterior corpus, B2: mid-

Since 1966, more than 12,000 research articles have been published on the epididymis. These articles, while addressing the various aspects of the epididymis from several points of view, all agree that the epididymis is crucial for the preparation of the spermatozoa prior to fertilization. It is well known that the proteins and small molecules secreted by the epididymal epithelium into the lumen interact with the transiting spermatozoa and directly or indirectly affect the spermatozoa surface; these proteins and small molecules function as signaling molecules to induce activity in the other epididymal proteins (Gatti et al., 2004). A significant number of these molecules will be taken up by the spermatozoa. Several of the acrosome molecules, which were previously formed during spermiogenesis and are involved in the acrosome reaction or in the sperm-zona pellucida interaction and sperm-egg fusion, will be gradually rearranged and compartmentalized in a stage-specific manner

Through research, we have learned a substantial amount about the function and the biochemical properties of the epididymal structure. However, there are still gaps in nearly all aspects of the research. Studying the complexity of the cellular properties, the spatial and temporal organization of protein syntheses and secretion and the dynamic interactions

corpus, B3: posterior corpus, C1: anterior cauda, C2: posterior cauda).

during sperm maturation (França et al., 2005).

In mammals, the genetic sex of the embryo is determined mainly by the presence or absence of a single gene on the Y chromosome, the SRY, which is required to initiate male-specific pathways and to repress female-specific pathways of development (Moore & Persaud, 2003).  The gonads (testicles or ovaries), the genital ducts (Wolffian or Müllerian) and the urogenital sinus of both sexes emerge from the morphologically undifferentiated primordia. We can then assume that the genes involved in the establishment of these primordia are the same for both sexes. During development, the first morphological sex difference appear in the gonads of male embryos, followed by the genital ducts, and finally, they appear in the urogenital sinus (Larios & Mendoza, 2001).

In humans, the genital ducts arise from the intermediate mesoderm. Shortly after the formation of the Wolffian ducts, the adjacent Müllerian ducts are formed. In males, sexual differentiation develops through regression of the Müllerian ducts and the development of the Wolffian ducts into multiple reproductive organs, such as the epididymis, the vas deferens ducts and the the seminal vesicles. In females, the Wolffian ducts regress and the Müllerian ducts differentiate to form the uterus, the fallopian tubes, and the proximal vagina (Hannema & Hughes, 2007; Kobayashi & Behringer, 2003).

In the male tract, Müllerian duct regression, which depends on the testicular Sertoli cells, is mediated by the Y chromosome genes and is triggered by a mechanism that is not well understood. The Sertoli cells produce and secrete an anti-Müllerian substance, a nonsteroidal hormone. Under the control of human chorionic gonadotrophin (hCG), a placental gonadotrophin, the Leydig cells then differentiate and produce the androgens that will positively regulate the ipsilateral Wolffian duct in a paracrine way. These hormonal combinations induce the cranial Wolffian duct to become highly convoluted and to differentiate into the epididymis.

The major morphogenic event during Wolffian duct/epididymal duct development is the elongation and coiling of the duct (Hannema & Hughes, 2007). The process of elongation is likely a product of potential mechanisms such as cell proliferation coupled with directed cell rearrangements, along with the interactions between the Wolffian duct ephitelium and the surrounding mesenchyme cells (Hinton et al., 2011).

The Epididymis: Embryology, Structure, Function and Its Role in Fertilization and Infertility 45

Fig. 3. A schematic diagram that describes the differentiation of the mouse epididymal epithelium from birth until adulthood. A to F indicate the different types of cells in the

By birth, the presumptive areas of the initial segment, caput and corpus have experienced considerable coiling compared with the epididymal cauda (Figure 4). Within the first few days after birth, the individual conjunctive septum initiates the anatomical segmentation of epididymis, along with the considerable growth of the duct. The epithelium is still undifferentiated and characterized by columnar cells. The histological changes do not begin until the first stages of early infancy with the appearance of halo cells (Robaire et al., 2000, 2006) (Figure 3). The first phase begins, then, with the undifferentiated period and ends with the first event of the differentiation period, which is defined by the appearance of halo cells.

The epididymal epithelium differentiates into the pseudostratified epithelium, which contains principal, basal, apical, narrow and clear cells (Figure 3). Epithelial cell differentiation is completed during puberty when the highest rate of cell division and epididymal expansion occurs. This period of expansion describes the continued growth of

epithelium of the epididymis.

**3.1 The first phase: From birth to early infancy**

**3.2 The second phase: From early infancy to adulthood**

The Wolffian ducts also induce the formation of the mesonephric tubules in the mesonephric mesenchyme, which extend to the epithelial cells of the gonad and subsequently differentiate into efferent ductules. These ductules open into the epididymis (Figure 2). Distal to the epididymis, the Wolffian ducts acquire a thicker investment of smooth muscle and become the ductus deferens (Moore & Persaud, 2003; Hannema & Hughes, 2007).

Fig. 2. The intra-uterine development of the epididymis. A) A sagittal section of a mouse embryo showing the relative locations of the developing urogenital structures. B) The testicular production and secretion of testosterone positively regulates the Wolffian ducts. The ducts then induce the formation of the mesonephric tubules in the mesonephric mesenchyme that extend to the epithelial cells of the gonad. C) The cords arising from the apical mesonephric tubules form the efferent ducts and fuse with the adjacent ducts to form the rete testis. The coiling of the initial segment of the efferent duct is not shown here, but it proceeds independently of the coiling of the main epididymal duct shown in D, E and F. D) The coiling shifts from the proximal to the distal duct in a temporal fashion. E) The initial stages of coiling are planar, meaning that the coiling is at the two-dimensional level. F) The three-dimensional coiling proceeds in a caput-to-cauda direction and is completed in the early postnatal period.

#### **3. The postnatal development of the epididymis**

While cell proliferation in the Wolffian duct appears to be dependent on the presence of androgens and mesenchymal factors during prenatal development, lumicrine factors produced by the testicles play an additional role during postnatal development (Hinton et al., 2011). Lumicrine factors are molecules (such as androgens, growth factors and enzymes) whose effect on the secretory activity of the epithelial cells of the epididymis and in the epididymis spermatozoa directly participates in the process of epididymal maturation (Lan et al., 1998; Robaire et al., 2006). In general, the postnatal development of the mammalian epididymis can be divided into three periods that take place across two developmental phases (Figure 3).

The Wolffian ducts also induce the formation of the mesonephric tubules in the mesonephric mesenchyme, which extend to the epithelial cells of the gonad and subsequently differentiate into efferent ductules. These ductules open into the epididymis (Figure 2). Distal to the epididymis, the Wolffian ducts acquire a thicker investment of smooth muscle and become the ductus deferens (Moore & Persaud, 2003; Hannema &

Fig. 2. The intra-uterine development of the epididymis. A) A sagittal section of a mouse embryo showing the relative locations of the developing urogenital structures. B) The testicular production and secretion of testosterone positively regulates the Wolffian ducts. The ducts then induce the formation of the mesonephric tubules in the mesonephric mesenchyme that extend to the epithelial cells of the gonad. C) The cords arising from the apical mesonephric tubules form the efferent ducts and fuse with the adjacent ducts to form the rete testis. The coiling of the initial segment of the efferent duct is not shown here, but it proceeds independently of the coiling of the main epididymal duct shown in D, E and F. D) The coiling shifts from the proximal to the distal duct in a temporal fashion. E) The initial stages of coiling are planar, meaning that the coiling is at the two-dimensional level. F) The three-dimensional coiling proceeds in a caput-to-cauda direction and is completed in the

While cell proliferation in the Wolffian duct appears to be dependent on the presence of androgens and mesenchymal factors during prenatal development, lumicrine factors produced by the testicles play an additional role during postnatal development (Hinton et al., 2011). Lumicrine factors are molecules (such as androgens, growth factors and enzymes) whose effect on the secretory activity of the epithelial cells of the epididymis and in the epididymis spermatozoa directly participates in the process of epididymal maturation (Lan et al., 1998; Robaire et al., 2006). In general, the postnatal development of the mammalian epididymis can be divided into three periods that take place across two developmental

Hughes, 2007).

early postnatal period.

phases (Figure 3).

**3. The postnatal development of the epididymis**

Fig. 3. A schematic diagram that describes the differentiation of the mouse epididymal epithelium from birth until adulthood. A to F indicate the different types of cells in the epithelium of the epididymis.

#### **3.1 The first phase: From birth to early infancy**

By birth, the presumptive areas of the initial segment, caput and corpus have experienced considerable coiling compared with the epididymal cauda (Figure 4). Within the first few days after birth, the individual conjunctive septum initiates the anatomical segmentation of epididymis, along with the considerable growth of the duct. The epithelium is still undifferentiated and characterized by columnar cells. The histological changes do not begin until the first stages of early infancy with the appearance of halo cells (Robaire et al., 2000, 2006) (Figure 3). The first phase begins, then, with the undifferentiated period and ends with the first event of the differentiation period, which is defined by the appearance of halo cells.

#### **3.2 The second phase: From early infancy to adulthood**

The epididymal epithelium differentiates into the pseudostratified epithelium, which contains principal, basal, apical, narrow and clear cells (Figure 3). Epithelial cell differentiation is completed during puberty when the highest rate of cell division and epididymal expansion occurs. This period of expansion describes the continued growth of

The Epididymis: Embryology, Structure, Function and Its Role in Fertilization and Infertility 47

epithelium and the luminal protein secretion within the three main epididymal segments can be further subdivided into several regions (França et al., 2005; Robaire et al., 2006; Turner, 2008). In general, the five physiological regions are distinguished as follows: (i) the proximal caput or the initial segment, (ii) the middle caput, (iii) the distal caput and the proximal corpus, (iv) the distal corpus and (v) the cauda (Dacheux et al., 2005; Turner, 2008). Note that the proposed physiological regions are slightly different from the anatomical

The differential response of the segments to androgen withdrawal, stress and aging indicates that each region represents discrete regulatory units (Jervis & Robaire, 2001). The epididymis is more than a uniform channel that transports and stores spermatozoa. The maturation and storage of the spermatozoa depends upon the epithelial tight and adhering junctions. They are important in maintaining the integrity of the epididymal epithelium and in the formation of the blood epididymal barrier. Tight junctions between the adjacent epididymal epithelial cells form the blood-epididymal barrier and restrict the passage of a number of ions, solutes, and macromolecules through the epididymal ephitelium. This barrier also serves as an extension of the blood-testis barrier. The spermatozoa are immunogenic and contain proteins on their surfaces that would be recognized as nonself if they leave the epididymis (Robaire & Hermo, 1988; Dacheux

Histological characteristics allow for the easy identification of the anterior and posterior extremities of the mammalian epididymis. The thickness of the epididymal epithelium varies with the thickest portion in the proximal caput and the thinnest in the caudal region (Figure 5). Conversely, the luminal diameter and the thickness of the peritubular smooth muscle increases from the proximal to the distal regions (Lasserre et al., 2001; Toshimori, 2003). Few sperm are found in the initial segment, but a large mass of sperm aggregates are located in the cauda (Yanagimachi et al., 1985; Cornwall, 2009). In all of these segments, the epididymal duct is lined with an epithelium composed of principal and basal cells. Other cells, such as apical, narrow, clear and halo cells, are also present in this duct in a segment-

Fig. 5. Photomicrographs showing the histological regionalization of the A) initial segment and caput separated by the connective tissue septa, B) the corpus and C) the cauda of the mouse epididymis. The thickness of the epididymal epithelium varies from the thickest portion in the proximal caput to the thinnest in the caudal region. Conversely, the luminal diameter and the thickness of the peritubular smooth muscle increases from the proximal to

regions (Figure 1).

et al., 2005).

specific manner (Figure 6).

the distal regions. Scale bar: 50µm.

the duct and the appearance of spermatozoa in the epididymal lumen. The establishment of the regionalized protein secretion takes place progressively coupled to the consolidation of an anatomical epididymal segmentation (Figure 4). The differentiation and regionalization are related to the different stages of testicular maturation, the steroidogenic activity of the Leydig cells, the androgen dependence of the epididymis itself and the lumicrine factors (De Miguel et al., 1998; Robaire et al., 2000; Dacheux et al., 2005; Cornwall, 2009; Robaire et al., 2006). Then the second phase starts after halo cell differentiation and ends in puberty.

Fig. 4. Photomicrographs of a mouse epididymis. A) At birth, the undifferentiated period shows a poorly coiling duct and little regionalization. B) A well-formed adult epididymis in expansion with a completely coiled and regionalized duct. Scale bar: A) 100µm. B) 1100µm.

#### **4. Structural features**

In the testes, the seminiferous tubules converge to form the rete testis, which in turn gives rise to the efferent ducts. The number of tubules contained in the efferent ducts varies depending on the species and on how the tubules converge to form the single coiled duct of the epididymis. The epididymal tubular lumen is continuous with the lumina of the efferent ducts and comes to an end in the vas deferens, which has a thick layer of muscle. The urethra is the final extension of the vas deferens and communicates with the outside of the body.

#### **4.1 Macroscopic features**

The mammalian epididymis is an elongated coiled duct suspended within the mesorchium and firmly or loosely bound to the testicular tunica albuginea. The gross aspect of the epididymis allows the identification of the different segments (Figure 1), which are comprised of the proximal region (the initial segment and the caput), the corpus and the distal cauda (Robaire et al., 2006; Turner, 2008). In all mammalian species, each region of the epididymis is further organized into lobules that are separated by the connective tissue septa. These septa not only serve as internal support for the organ but are also thought to provide a functional separation between the lobules that allows for the selective expression of genes and proteins within the individual lobules (Kirchhoff, 1999; Cornwall, 2009).

#### **4.2 Microscopic features**

The epididymis also contains region-specific and cell-specific functions specifically located within the epithelium of a given segment. The highly specific regionalization of the

the duct and the appearance of spermatozoa in the epididymal lumen. The establishment of the regionalized protein secretion takes place progressively coupled to the consolidation of an anatomical epididymal segmentation (Figure 4). The differentiation and regionalization are related to the different stages of testicular maturation, the steroidogenic activity of the Leydig cells, the androgen dependence of the epididymis itself and the lumicrine factors (De Miguel et al., 1998; Robaire et al., 2000; Dacheux et al., 2005; Cornwall, 2009; Robaire et al., 2006). Then the second phase starts after halo cell differentiation and ends in puberty.

Fig. 4. Photomicrographs of a mouse epididymis. A) At birth, the undifferentiated period shows a poorly coiling duct and little regionalization. B) A well-formed adult epididymis in expansion with a completely coiled and regionalized duct. Scale bar: A) 100µm. B) 1100µm.

In the testes, the seminiferous tubules converge to form the rete testis, which in turn gives rise to the efferent ducts. The number of tubules contained in the efferent ducts varies depending on the species and on how the tubules converge to form the single coiled duct of the epididymis. The epididymal tubular lumen is continuous with the lumina of the efferent ducts and comes to an end in the vas deferens, which has a thick layer of muscle. The urethra is the final extension of the vas deferens and communicates with the outside

The mammalian epididymis is an elongated coiled duct suspended within the mesorchium and firmly or loosely bound to the testicular tunica albuginea. The gross aspect of the epididymis allows the identification of the different segments (Figure 1), which are comprised of the proximal region (the initial segment and the caput), the corpus and the distal cauda (Robaire et al., 2006; Turner, 2008). In all mammalian species, each region of the epididymis is further organized into lobules that are separated by the connective tissue septa. These septa not only serve as internal support for the organ but are also thought to provide a functional separation between the lobules that allows for the selective expression of genes and proteins within the individual lobules (Kirchhoff, 1999;

The epididymis also contains region-specific and cell-specific functions specifically located within the epithelium of a given segment. The highly specific regionalization of the

**4. Structural features**

**4.1 Macroscopic features**

of the body.

Cornwall, 2009).

**4.2 Microscopic features**

epithelium and the luminal protein secretion within the three main epididymal segments can be further subdivided into several regions (França et al., 2005; Robaire et al., 2006; Turner, 2008). In general, the five physiological regions are distinguished as follows: (i) the proximal caput or the initial segment, (ii) the middle caput, (iii) the distal caput and the proximal corpus, (iv) the distal corpus and (v) the cauda (Dacheux et al., 2005; Turner, 2008). Note that the proposed physiological regions are slightly different from the anatomical regions (Figure 1).

The differential response of the segments to androgen withdrawal, stress and aging indicates that each region represents discrete regulatory units (Jervis & Robaire, 2001). The epididymis is more than a uniform channel that transports and stores spermatozoa. The maturation and storage of the spermatozoa depends upon the epithelial tight and adhering junctions. They are important in maintaining the integrity of the epididymal epithelium and in the formation of the blood epididymal barrier. Tight junctions between the adjacent epididymal epithelial cells form the blood-epididymal barrier and restrict the passage of a number of ions, solutes, and macromolecules through the epididymal ephitelium. This barrier also serves as an extension of the blood-testis barrier. The spermatozoa are immunogenic and contain proteins on their surfaces that would be recognized as nonself if they leave the epididymis (Robaire & Hermo, 1988; Dacheux et al., 2005).

Histological characteristics allow for the easy identification of the anterior and posterior extremities of the mammalian epididymis. The thickness of the epididymal epithelium varies with the thickest portion in the proximal caput and the thinnest in the caudal region (Figure 5). Conversely, the luminal diameter and the thickness of the peritubular smooth muscle increases from the proximal to the distal regions (Lasserre et al., 2001; Toshimori, 2003). Few sperm are found in the initial segment, but a large mass of sperm aggregates are located in the cauda (Yanagimachi et al., 1985; Cornwall, 2009). In all of these segments, the epididymal duct is lined with an epithelium composed of principal and basal cells. Other cells, such as apical, narrow, clear and halo cells, are also present in this duct in a segmentspecific manner (Figure 6).

Fig. 5. Photomicrographs showing the histological regionalization of the A) initial segment and caput separated by the connective tissue septa, B) the corpus and C) the cauda of the mouse epididymis. The thickness of the epididymal epithelium varies from the thickest portion in the proximal caput to the thinnest in the caudal region. Conversely, the luminal diameter and the thickness of the peritubular smooth muscle increases from the proximal to the distal regions. Scale bar: 50µm.

The Epididymis: Embryology, Structure, Function and Its Role in Fertilization and Infertility 49

they are in close association with the overlying principal cells or other basal cells through the presence of cytoplasmatic extensions (Robaire et al., 2000; Cornwall, 2009). Because of this contact with the basement membrane, basal cells form an extensive cellular sheet surrounding the epididymal epithelium (Robaire et al., 2006; Cornwall, 2009). Although basal cells are extratubular in origin, some findings have suggested that the cells may have a role within the processes of the epithelial immune system and in the regulation of electrolytes by principal cells. However, the exact functions of these cells are not yet clear

Apical cells (Figure 3D) comprise approximately 10% of the total epithelial population in the initial segment but only approximately 1% of the total epithelial population in the cauda of the epididymis (Adamali & Hermo, 1996). They are clearly defined by the many mitochondria in the apical cytoplasm, the few microvilli at the luminal border and a nucleus that is located in the upper half of the cell cytoplasm (Adamali & Hermo, 1996; Robaire et al., 2006). These cells are related to sperm quiescence and to the regulation of the pH in the lumen through the

Narrow cells (Figure 3E) are the slender elongated cells. They increase from 3% of the total epithelial population in the initial segment to 6% of the total epithelial population in the corpus. These cells presents numerous C-shaped vesicles and mitochondria with a small flattened nucleus located in the upper half of the cell cytoplasm. The structural features of both apical and narrow cells suggest that these cells are involved in the process of intracellular transport between the lumen and the epithelial cells, in the degradation of specific proteins and carbohydrates within their lysosomes and in protecting spermatozoa from a changing environment of harmful electrophiles (Adamali & Hermo, 1996; Robaire et al., 2006). They also differ dramatically from the neighboring principal cells and display region-specific expression of proteins, such as the glutathione S-transferases and lysosomal

Clear cells (Figure 3F), along with halo cells, constitute fewer than 5% of the total epithelial cell population. Clear cells are equally distributed through the caput, the corpus and the cauda segments. The dark-stained nucleus of these cells is surrounded by the pale-staining cytoplasm. They are also present in all levels of the epididymal epithelium. Clear cells are also endocytic cells and may be responsible for the clearance of proteins from the epididymal lumen. They normally take up the contents of the cytoplasmic droplets released by the spermatozoa as they transit through the duct (Hermo et al., 1994, 2005; Robaire et al., 2006).

Halo cells (Figure 3A) are usually located in the base of the ephitelium where it does not touch the basement membrane. These cells contain variable numbers of dense core granules.

production of enzymes of the carbonic anhydrase family (Hermo et al., 2005).

(Robaire et al., 2006).

**4.2.3 Apical cells**

**4.2.4 Narrow cells**

**4.2.5 Clear cells**

**4.2.6 Halo cells**

enzymes (Adamali & Hermo, 1996).

Fig. 6. A schematic cross section of epididymis showing the organization of the major cell types in the epithelium of the epididymal duct as observed through a light microscope. A generic epididymal compartment with the relative position and distribution of all cell types found throughout the epithelium is illustrated. The thinnest coat of organized smooth muscle is in the interstitial tissue surrounding the duct and is characteristic of the anterior regions of the epididymis. Note the spermatozoa in the center of the epididymal lumen.

#### **4.2.1 Principal cells**

Principal cells (Figure 3B) are the most abundant and extensively studied cell type found in the epididymal epithelium. Principal cells constitute approximately 80% of the total epithelial cell population in the initial segment. The number of principal cells gradually decreases to 65% of the total epithelial cell population in the cauda epididymis (Robaire & Hermo, 1988). These columnar cells present prominent stereocilia and extend into the lumen. Ultrastructurally, the supranuclear region of this cell type contains large stacks of Golgi saccules, mitochondria, multivesicular bodies and apical dilated membranous elements, while the infranuclear region is densely packed with rough endoplasmic reticulum (Robaire et al., 2000, Dacheux et al., 2005). Principal cells are responsible for the bulk of the proteins that are secreted into the lumen and are directly involved in the control of luminal protein concentrations. They frequently exhibit blebs of cytoplasm emanating from their apical cell surface. These cells also form tight junctions with one another, and as such, form the blood-epididymis barrier (Robaire et al., 2006; Cornwall, 2009).

#### **4.2.2 Basal cells**

Basal cells (Figure 3C) are the second most abundant cell type found in the epididymal epithelium, constituting 15-20% of the total epithelial cell population of the epididymis. They are triangular and flat cells and they reside in the base of the epithelium. Basal cells cannot access the luminal compartment. They have elongated or round shaped nuclei, and they are in close association with the overlying principal cells or other basal cells through the presence of cytoplasmatic extensions (Robaire et al., 2000; Cornwall, 2009). Because of this contact with the basement membrane, basal cells form an extensive cellular sheet surrounding the epididymal epithelium (Robaire et al., 2006; Cornwall, 2009). Although basal cells are extratubular in origin, some findings have suggested that the cells may have a role within the processes of the epithelial immune system and in the regulation of electrolytes by principal cells. However, the exact functions of these cells are not yet clear (Robaire et al., 2006).

#### **4.2.3 Apical cells**

48 Embryology – Updates and Highlights on Classic Topics

Fig. 6. A schematic cross section of epididymis showing the organization of the major cell types in the epithelium of the epididymal duct as observed through a light microscope. A generic epididymal compartment with the relative position and distribution of all cell types found throughout the epithelium is illustrated. The thinnest coat of organized smooth muscle is in the interstitial tissue surrounding the duct and is characteristic of the anterior regions of the epididymis. Note the spermatozoa in the center of the epididymal lumen.

Principal cells (Figure 3B) are the most abundant and extensively studied cell type found in the epididymal epithelium. Principal cells constitute approximately 80% of the total epithelial cell population in the initial segment. The number of principal cells gradually decreases to 65% of the total epithelial cell population in the cauda epididymis (Robaire & Hermo, 1988). These columnar cells present prominent stereocilia and extend into the lumen. Ultrastructurally, the supranuclear region of this cell type contains large stacks of Golgi saccules, mitochondria, multivesicular bodies and apical dilated membranous elements, while the infranuclear region is densely packed with rough endoplasmic reticulum (Robaire et al., 2000, Dacheux et al., 2005). Principal cells are responsible for the bulk of the proteins that are secreted into the lumen and are directly involved in the control of luminal protein concentrations. They frequently exhibit blebs of cytoplasm emanating from their apical cell surface. These cells also form tight junctions with one another, and as

Basal cells (Figure 3C) are the second most abundant cell type found in the epididymal epithelium, constituting 15-20% of the total epithelial cell population of the epididymis. They are triangular and flat cells and they reside in the base of the epithelium. Basal cells cannot access the luminal compartment. They have elongated or round shaped nuclei, and

such, form the blood-epididymis barrier (Robaire et al., 2006; Cornwall, 2009).

**4.2.1 Principal cells**

**4.2.2 Basal cells**

Apical cells (Figure 3D) comprise approximately 10% of the total epithelial population in the initial segment but only approximately 1% of the total epithelial population in the cauda of the epididymis (Adamali & Hermo, 1996). They are clearly defined by the many mitochondria in the apical cytoplasm, the few microvilli at the luminal border and a nucleus that is located in the upper half of the cell cytoplasm (Adamali & Hermo, 1996; Robaire et al., 2006). These cells are related to sperm quiescence and to the regulation of the pH in the lumen through the production of enzymes of the carbonic anhydrase family (Hermo et al., 2005).

#### **4.2.4 Narrow cells**

Narrow cells (Figure 3E) are the slender elongated cells. They increase from 3% of the total epithelial population in the initial segment to 6% of the total epithelial population in the corpus. These cells presents numerous C-shaped vesicles and mitochondria with a small flattened nucleus located in the upper half of the cell cytoplasm. The structural features of both apical and narrow cells suggest that these cells are involved in the process of intracellular transport between the lumen and the epithelial cells, in the degradation of specific proteins and carbohydrates within their lysosomes and in protecting spermatozoa from a changing environment of harmful electrophiles (Adamali & Hermo, 1996; Robaire et al., 2006). They also differ dramatically from the neighboring principal cells and display region-specific expression of proteins, such as the glutathione S-transferases and lysosomal enzymes (Adamali & Hermo, 1996).

#### **4.2.5 Clear cells**

Clear cells (Figure 3F), along with halo cells, constitute fewer than 5% of the total epithelial cell population. Clear cells are equally distributed through the caput, the corpus and the cauda segments. The dark-stained nucleus of these cells is surrounded by the pale-staining cytoplasm. They are also present in all levels of the epididymal epithelium. Clear cells are also endocytic cells and may be responsible for the clearance of proteins from the epididymal lumen. They normally take up the contents of the cytoplasmic droplets released by the spermatozoa as they transit through the duct (Hermo et al., 1994, 2005; Robaire et al., 2006).

#### **4.2.6 Halo cells**

Halo cells (Figure 3A) are usually located in the base of the ephitelium where it does not touch the basement membrane. These cells contain variable numbers of dense core granules.

The Epididymis: Embryology, Structure, Function and Its Role in Fertilization and Infertility 51

of T, is involved in the development of the prostate and the external genitalia. Although T is the predominantly active androgen during the first phase of the postnatal development of the epididymis, it is the effects of DHT that are important in the epididymal fluid of the mature epididymis. DHT can be produced locally in the epididymis by principal cells and is primarily found in the initial segment of the duct (Dacheux et al., 2005; França et al., 2005;

The actions of both T and DHT are initiated through the intracellular receptor known as the androgen receptor (AR). DHT is the more potent androgen of the two. The AR is found in all male reproductive organs and can be stimulated by either T or its more potent metabolite, DHT. The binding of either T or DHT to the AR may regulate distinct androgenic effects in target tissues. Clinical syndromes, such as androgen insensitivity (AIS), illustrate the differential actions of T and DHT (Umar et al., 2003). AR expression in the developing male genital tract occurs in a strict temporal pattern. It is first detected in the mesenchymal cells, then in the epithelial cells and then in both the epithelial and stromal compartments of the

The initiation of androgen-dependent differentiation of the Wolffian duct system into epididymis occurs before epithelial cells express a detectable level of the AR protein. In this phase of development, the mesenchymal cells are important androgen targets that elicit androgenic effects in the epithelial cells via paracrine factors and mesenchymal-epithelial interactions (Umar et al., 2003). Genetic mutations in the AR or treatment with AR antagonists during the male embryonic stage results in the regression of the Wolffian ducts

During postnatal development, the luminal secretion of androgens is essential for the maintenance of epithelial cell identity (O´Hara et al., 2011), and for the normal development and function of the stromal cells (Nitta et al., 1993; Robaire et al., 2000; Hess et al., 2001; O'Hara et al., 2011). Both the regionalized differentiation of the epididymis and the variation in the luminal fluid composition take place under the control of androgens (Toshimori, 2003). The production and secretion of at least half of the epididymal proteins, including those later incorporated by the spermatozoa in transit, are under androgenic control. Androgenic control may act positively or negatively, depending on the varying levels of sensitivity (Tezon et al., 1985; Ellerman et al., 1998; Robaire et al., 2000, Dachuex et al. 2005;

In addition to testosterone and other androgenic-derived metabolites, estrogen has been reported to target epididymal epithelial cells (Hess et al., 2001, 2011). The presence of two estrogen receptors (ESR) types in the head of the epididymis, as well as in other regions of the epididymis, has been well documented. Their expression appears to be isotype-, species-, and cell-specific (Hess et al., 2001, 2011). Recent studies have shown that the luminal fluid reabsorption that occurs in the efferent ductules and in the initial segment of the epididymis is regulated by estrogen. The estrogen present in the epididymis also regulates the transport of fluid through the duct and is responsible for increasing the concentration of sperm as they enter the caput of the epididymis (França et al., 2005; Hess et al., 2011). Estrogen assists in the maintenance of a differentiated epithelial morphology,

Robaire & Henderson, 2006).

Robaire & Henderson, 2006).

**5.1.2 Estrogen**

epididymis (Umar et al., 2003; O'Hara et al., 2011).

and an absence of the epididymis in adult males (O'Hara et al., 2011).

They develop in the immune system from a combination of B and T lymphocytes and monocytes (Dacheux et al., 2005; Robaire et al., 2006).

#### **4.2.7 Cell interactions in the epididymal duct**

The cell types described above are active in the processes of epididymal function, such as protein secretion and absorption (principal cells); endocytosis (clear and apical cells); the secretory activities responsible for the acidification of the luminal fluid (clear cells and narrow cells); immune defense; phagocytosis (halo cells); and the production of antioxidants (basal cells) (Robaire et al., 2000; França et al., 2005). It is important to understand that each cell type may express different proteins within the distinct epididymal regions. This indicates that the cells perform different functions according to their location, and that the specificity of epididymal secretions is progressively established with age (Robaire et al., 2000, 2006). This information confirms the high degree of regionalization involved in the activity of the epididymis. This epididymal regionalization, which is attributed to the diverse patterns of gene expression, is critical to the formation and maintenance of the functions of the epididymal duct (Suzuki et al., 2004).

#### **5. The maintenance of the epididymis**

In mammals, the development and maintenance of a fully differentiated epididymal epithelium is dependent on a combination of factors that provide an ideal site where the spermatozoa undergo a series of morphological, biochemical and physiological changes. During this process of epididymal maturation, the spermatozoa move along the ducts in a fluid that dynamically evolves through the processes of absorption and secretion by the epithelial cells, androgens, as well as through lumicrine factors from the testis (Cornwall, 2009).

#### **5.1 Hormones**

Androgens play a crucial role in the development of the male reproductive organs, such as the testis, the epididymis, the vas deferens, the seminal vesicle, the prostate and the penis. The role of androgens is an important topic in the study of puberty, male fertility and male sexual function. The effects of androgen withdrawal have been well established through the experimental model of orchiectomy. A decrease in the weight of the epididymis has been commonly observed in animals that have had their testicles removed. In these cases, androgen replacement, even at supraphysiological levels, only partially restored the weight of the epididymis. The removal of the testicles caused the loss of androgens, but it is clear that this approach affected estrogen levels and other testicular factors that may affect the maintenance of epididymis (Robaire et al., 2000).

#### **5.1.1 Androgen: Testosterone and dihydrotestosterone**

The formation and function of the epididymis is androgen-dependent. The principal androgen, testosterone (T), is essential for the development of the internal sex organs and is derived from the Wolffian duct system, which consists of the epididymis, the vas deferens, and the seminal vesicle (Umar et al., 2003). Dihydrotestosterone (DHT), the 5α-reduced form of T, is involved in the development of the prostate and the external genitalia. Although T is the predominantly active androgen during the first phase of the postnatal development of the epididymis, it is the effects of DHT that are important in the epididymal fluid of the mature epididymis. DHT can be produced locally in the epididymis by principal cells and is primarily found in the initial segment of the duct (Dacheux et al., 2005; França et al., 2005; Robaire & Henderson, 2006).

The actions of both T and DHT are initiated through the intracellular receptor known as the androgen receptor (AR). DHT is the more potent androgen of the two. The AR is found in all male reproductive organs and can be stimulated by either T or its more potent metabolite, DHT. The binding of either T or DHT to the AR may regulate distinct androgenic effects in target tissues. Clinical syndromes, such as androgen insensitivity (AIS), illustrate the differential actions of T and DHT (Umar et al., 2003). AR expression in the developing male genital tract occurs in a strict temporal pattern. It is first detected in the mesenchymal cells, then in the epithelial cells and then in both the epithelial and stromal compartments of the epididymis (Umar et al., 2003; O'Hara et al., 2011).

The initiation of androgen-dependent differentiation of the Wolffian duct system into epididymis occurs before epithelial cells express a detectable level of the AR protein. In this phase of development, the mesenchymal cells are important androgen targets that elicit androgenic effects in the epithelial cells via paracrine factors and mesenchymal-epithelial interactions (Umar et al., 2003). Genetic mutations in the AR or treatment with AR antagonists during the male embryonic stage results in the regression of the Wolffian ducts and an absence of the epididymis in adult males (O'Hara et al., 2011).

During postnatal development, the luminal secretion of androgens is essential for the maintenance of epithelial cell identity (O´Hara et al., 2011), and for the normal development and function of the stromal cells (Nitta et al., 1993; Robaire et al., 2000; Hess et al., 2001; O'Hara et al., 2011). Both the regionalized differentiation of the epididymis and the variation in the luminal fluid composition take place under the control of androgens (Toshimori, 2003). The production and secretion of at least half of the epididymal proteins, including those later incorporated by the spermatozoa in transit, are under androgenic control. Androgenic control may act positively or negatively, depending on the varying levels of sensitivity (Tezon et al., 1985; Ellerman et al., 1998; Robaire et al., 2000, Dachuex et al. 2005; Robaire & Henderson, 2006).

#### **5.1.2 Estrogen**

50 Embryology – Updates and Highlights on Classic Topics

They develop in the immune system from a combination of B and T lymphocytes and

The cell types described above are active in the processes of epididymal function, such as protein secretion and absorption (principal cells); endocytosis (clear and apical cells); the secretory activities responsible for the acidification of the luminal fluid (clear cells and narrow cells); immune defense; phagocytosis (halo cells); and the production of antioxidants (basal cells) (Robaire et al., 2000; França et al., 2005). It is important to understand that each cell type may express different proteins within the distinct epididymal regions. This indicates that the cells perform different functions according to their location, and that the specificity of epididymal secretions is progressively established with age (Robaire et al., 2000, 2006). This information confirms the high degree of regionalization involved in the activity of the epididymis. This epididymal regionalization, which is attributed to the diverse patterns of gene expression, is critical to the formation and maintenance of the

In mammals, the development and maintenance of a fully differentiated epididymal epithelium is dependent on a combination of factors that provide an ideal site where the spermatozoa undergo a series of morphological, biochemical and physiological changes. During this process of epididymal maturation, the spermatozoa move along the ducts in a fluid that dynamically evolves through the processes of absorption and secretion by the epithelial cells, androgens, as well as through lumicrine factors from the

Androgens play a crucial role in the development of the male reproductive organs, such as the testis, the epididymis, the vas deferens, the seminal vesicle, the prostate and the penis. The role of androgens is an important topic in the study of puberty, male fertility and male sexual function. The effects of androgen withdrawal have been well established through the experimental model of orchiectomy. A decrease in the weight of the epididymis has been commonly observed in animals that have had their testicles removed. In these cases, androgen replacement, even at supraphysiological levels, only partially restored the weight of the epididymis. The removal of the testicles caused the loss of androgens, but it is clear that this approach affected estrogen levels and other testicular factors that may affect the

The formation and function of the epididymis is androgen-dependent. The principal androgen, testosterone (T), is essential for the development of the internal sex organs and is derived from the Wolffian duct system, which consists of the epididymis, the vas deferens, and the seminal vesicle (Umar et al., 2003). Dihydrotestosterone (DHT), the 5α-reduced form

monocytes (Dacheux et al., 2005; Robaire et al., 2006).

functions of the epididymal duct (Suzuki et al., 2004).

**5. The maintenance of the epididymis**

maintenance of epididymis (Robaire et al., 2000).

**5.1.1 Androgen: Testosterone and dihydrotestosterone**

testis (Cornwall, 2009).

**5.1 Hormones**

**4.2.7 Cell interactions in the epididymal duct**

In addition to testosterone and other androgenic-derived metabolites, estrogen has been reported to target epididymal epithelial cells (Hess et al., 2001, 2011). The presence of two estrogen receptors (ESR) types in the head of the epididymis, as well as in other regions of the epididymis, has been well documented. Their expression appears to be isotype-, species-, and cell-specific (Hess et al., 2001, 2011). Recent studies have shown that the luminal fluid reabsorption that occurs in the efferent ductules and in the initial segment of the epididymis is regulated by estrogen. The estrogen present in the epididymis also regulates the transport of fluid through the duct and is responsible for increasing the concentration of sperm as they enter the caput of the epididymis (França et al., 2005; Hess et al., 2011). Estrogen assists in the maintenance of a differentiated epithelial morphology,

The Epididymis: Embryology, Structure, Function and Its Role in Fertilization and Infertility 53

Fig. 7. A schematic illustration representing the distinct changes in the sperm surface throughout the stages of sperm maturation and epididymal cellular distribution throughout the epididymal epithelium. Histological characteristics allow for the

associate with the surface receptors and ions channels that are activated. Note the molecules that are also incorporated into the acrosomal vesicle. Some of these molecules

These molecules are indispensable to the specific recognition processes of the egg-

will persist until the capacitation reaction in the reproductive female tract.

spermatozoa interaction.

identification of the anterior and posterior extremities of the epididymis. The epididymal epithelium varies from its thickest section in the proximal caput to its thinnest section in the caudal region. The relative position and distribution of the main cell types are illustrated in each macroscopic structural region of the epididymis demonstrating that principal and basal cells are the two most abundant cell types throughout the entire epididymis. Detailed illustrations in the upper portion of the figure show the maturation of the spermatozoa, along with the incorporation of the molecules along the passage through the duct. Some molecules are bound, and later, may or may not disappear. Other molecules are incorporated into the cellular membrane of the plasma of the spermatozoa or

which means that it is absolutely necessary for the process of enhancing fertility in the male (Kobayashi & Behringer, 2003).

#### **5.2 Lumicrine factors**