**New Advances in Intracytoplasmic Sperm Injection (ICSI)**

Lodovico Parmegiani, Graciela Estela Cognigni and Marco Filicori *GynePro Medical Centers, Reproductive Medicine Unit, Bologna Italy* 

## **1. Introduction**

98 Advances in Embryo Transfer

Xu H, Beasley MD, Warren WD, van der Horst GT & McKay MJ. (2005). *Absence of mouse REC8 cohesin promotes synapsis of sister chromatids in meiosis*. Dev Cell 8:949-961. Zickler D. (2006). *From early homologue recognition to synaptonemal complex formation.* 

Zickler D & Kleckner N. (1998). *The leptotene-zygotene transition of meiosis*. Annu Rev Genet

*Chromosoma* 115:158-174.

32:619-697.

In these last twenty years, intracytoplasmic sperm injection (ICSI) has efficiently permitted the treatment of male factor infertility (Van Steirteghem et al., 1993); the direct injection of spermatozoa into ooplasm has allowed the embryologist to overcome low sperm motility, poor sperm-Zona Pellucida (ZP) binding, and defective acrosome reaction. Although ICSI has been successfully applied worldwide for several years, nevertheless we have no real knowledge regarding the hypothetical long term side effects on ICSI adults. In fact, some doubts about the safety of this technique can arise (Oehninger, 2011) due to the fact that with ICSI some check points of natural fertilization are bypassed and that some steps differ considerably from the physiological process; For instance, the introduction of the sperm tail into the ooplasm may cause sperm nuclear decondensation problems (Dozortsev et al., 1995; Markoulaki et al., 2007). It should be considered that ICSI may increase the risk of injecting spermatozoa with genetic or functional anomalies (Sakkas et al., 1997; Bonduelle et al., 2002; Marchesi & Feng, 2007; Schatten & Sun, 2009; Heytens et al., 2009; Navarro-Costa et al., 2010). For these reasons and to minimize any risk related to ICSI, any new advance in this procedure which can help the operator to restore some of the basic physiological checkpoints and to simulate the natural fertilization process should be welcome (Parmegiani et al., 2010a).

## **2. Hyaluronic acid (HA) and Zona Pellucida: Two important human fertilization checkpoints**

In nature, human oocytes are surrounded by:


These layers have to be penetrated by spermatozoa before they fuse with the oolemma.

In the human testis, during spermiogenesis, the elongated spermatids undergo cytoplasmic extrusion and plasma membrane remodelling which determines the formation of the HA and ZP receptors, essential for sperm penetration into oocyte.

New Advances in Intracytoplasmic Sperm Injection (ICSI) 101

 a plastic culture dish with microdots of HA hydrogel attached to the bottom of the dish (PICSI® Sperm Selection Device, MidAtlantic Diagnostic - Origio, Måløv, Denmark),

This new approach to ICSI with HA-bound spermatozoa, when using HA-viscous medium or HA-culture dishes, has been defined as "Physiologic ICSI" (Parmegiani et al., 2010a).

Since both these sperm-HA binding selection systems are easily available, efficient and approved for IVF use (Parmegiani et al., 2010 a; 2010 b; Mènèzo & Nicollet, 2004; Worrilow et al., 2007; 2010) IVF centres can choose the one best suited to their needs. The viscous medium requires a specific procedure of droplet preparation to optimize the selection of HA-bound spermatozoa (Parmegiani et al., 2010b); conversely, it is more versatile than PICSI as it can be used also on a glass-bottom culture dish for high magnification sperm evaluation: "physiologic IMSI" (Parmegiani et al., 2010 a) -*see also paragraph 5, IMSI*. On the other hand, PICSI HA-bound spermatozoa can be easily recognized even by non-trained embryologists.

PICSI dishes are conventional plastic culture dishes pre-prepared with 3 microdots of powdered HA. The powdered HA is re-hydrated by adding a 5 µL droplets of fresh culture medium to each of the three microdots. A 2 µL droplet with suspension of treated spermatozoa is then connected with a pipette tip to these culture medium droplets. The PICSI dish is incubated under oil; within 5 minutes the bound spermatozoa are attached by their head to the surface of the HA-microdots and are spinning around their head (Figure 1).

a viscous medium containing HA (Sperm Slow™, MediCult – Origio)

Fig. 1. Spermatozoa in PICSI dish (magnification 400 X)

Figure 1.

**3.1 PICSI procedure** 

At the end of spermiogenesis, different expression levels of two specific proteins seem to be related to sperm maturity, DNA integrity, chromosomal aneuploidy frequency and fertilizing potential. These two proteins are:


Mature spermatozoa have high HspA2 (Huszar et al., 2000) and low CK (Cayli et al., 2003). In contrast, spermatozoa with arrested maturity have low HspA2 expression, which may cause meiotic defects and probably chromosomal aneuploidies, in fact, mature spermatozoa show a reduction of more than five fold in aneupliody rate than immature ones (Jakab et al., 2005). Immature spermatozoa also have higher levels of CK (Huszar & Vigue, 1993); this high level of CK in immature spermatozoa is due to a sperm defect in terminal spermiogenesis when in normal development the surplus cytoplasm is extruded from the elongating spermatid as 'residual bodies' (Cayli et al., 2003). In contrast, arrested/ diminished maturity spermatozoa show a higher retention of cytoplasm with CK and other cytoplasmic enzymes, increased levels of lipid peroxidation and consequent DNA fragmentation, and abnormal sperm morphology. Due to the lack of their membrane remodelling, these immature spermatozoa have deficiency in the ZP and HA binding sites and for this reason they are not able to fertilize the oocyte naturally.

## **3. Physiologic HA ICSI**

In nature hyaluronic acid (HA), is involved in the mechanism of sperm selection because only mature spermatozoa which have extruded their specific receptors to bind to HA are able to reach the oocyte and fertilize it. The role of HA as "physiological selector" is also well recognized in-vitro. It has been demonstrated that the spermatozoa able to bind to HA in-vitro are those which have completed their plasma membrane remodelling, cytoplasmic extrusion and nuclear maturation (Cayli et al., 2003; Huszar et al., 2003; 2007). Furthermore, HA-bound spermatozoa have a better morphology (Prinosilova et al, 2009; Parmegiani et al., 2010a) and they show a reduced risk of being aneuploid (Jakab et al., 2005) or having fragmented DNA (Parmegiani et al., 2010a). Because of this, selection of spermatozoa by HA prior to ICSI helps to optimize the outcome of the treatment (Parmegiani et al., 2010 a, b) and also has a number of other advantages:


At very least, HA represents a more natural alternative for handling spermatozoa prior to ICSI than the synthetic plastic polyvinylpyrrolidone (PVP), which is routinely used to reduce sperm motility during ICSI procedure in the majority of AR centres and has been hypothesized to have toxic effects on oocytes (Jean et al., 1996; 2001).

A "home made" HA-sperm selection system can be simply produced in any IVF lab (Huszar et al., 2003, Nasr-Esfahani et al. 2008). However at the present time, two ready-to-use systems specially designed for sperm-HA binding selection are currently available:


This new approach to ICSI with HA-bound spermatozoa, when using HA-viscous medium or HA-culture dishes, has been defined as "Physiologic ICSI" (Parmegiani et al., 2010a).

Since both these sperm-HA binding selection systems are easily available, efficient and approved for IVF use (Parmegiani et al., 2010 a; 2010 b; Mènèzo & Nicollet, 2004; Worrilow et al., 2007; 2010) IVF centres can choose the one best suited to their needs. The viscous medium requires a specific procedure of droplet preparation to optimize the selection of HA-bound spermatozoa (Parmegiani et al., 2010b); conversely, it is more versatile than PICSI as it can be used also on a glass-bottom culture dish for high magnification sperm evaluation: "physiologic IMSI" (Parmegiani et al., 2010 a) -*see also paragraph 5, IMSI*. On the other hand, PICSI HA-bound spermatozoa can be easily recognized even by non-trained embryologists.

## **3.1 PICSI procedure**

100 Advances in Embryo Transfer

At the end of spermiogenesis, different expression levels of two specific proteins seem to be related to sperm maturity, DNA integrity, chromosomal aneuploidy frequency and

Mature spermatozoa have high HspA2 (Huszar et al., 2000) and low CK (Cayli et al., 2003). In contrast, spermatozoa with arrested maturity have low HspA2 expression, which may cause meiotic defects and probably chromosomal aneuploidies, in fact, mature spermatozoa show a reduction of more than five fold in aneupliody rate than immature ones (Jakab et al., 2005). Immature spermatozoa also have higher levels of CK (Huszar & Vigue, 1993); this high level of CK in immature spermatozoa is due to a sperm defect in terminal spermiogenesis when in normal development the surplus cytoplasm is extruded from the elongating spermatid as 'residual bodies' (Cayli et al., 2003). In contrast, arrested/ diminished maturity spermatozoa show a higher retention of cytoplasm with CK and other cytoplasmic enzymes, increased levels of lipid peroxidation and consequent DNA fragmentation, and abnormal sperm morphology. Due to the lack of their membrane remodelling, these immature spermatozoa have deficiency in the ZP and HA binding sites

In nature hyaluronic acid (HA), is involved in the mechanism of sperm selection because only mature spermatozoa which have extruded their specific receptors to bind to HA are able to reach the oocyte and fertilize it. The role of HA as "physiological selector" is also well recognized in-vitro. It has been demonstrated that the spermatozoa able to bind to HA in-vitro are those which have completed their plasma membrane remodelling, cytoplasmic extrusion and nuclear maturation (Cayli et al., 2003; Huszar et al., 2003; 2007). Furthermore, HA-bound spermatozoa have a better morphology (Prinosilova et al, 2009; Parmegiani et al., 2010a) and they show a reduced risk of being aneuploid (Jakab et al., 2005) or having fragmented DNA (Parmegiani et al., 2010a). Because of this, selection of spermatozoa by HA prior to ICSI helps to optimize the outcome of the treatment (Parmegiani et al., 2010 a, b)

in practical terms, HA-bound spermatozoa can be easily recovered using an injecting

HA-containing culture medium have no negative effects on post-injection zygote

Because of its natural origin HA can be metabolized by the oocyte (Balaban et al., 2003;

At very least, HA represents a more natural alternative for handling spermatozoa prior to ICSI than the synthetic plastic polyvinylpyrrolidone (PVP), which is routinely used to reduce sperm motility during ICSI procedure in the majority of AR centres and has been

A "home made" HA-sperm selection system can be simply produced in any IVF lab (Huszar et al., 2003, Nasr-Esfahani et al. 2008). However at the present time, two ready-to-use

systems specially designed for sperm-HA binding selection are currently available:

fertilizing potential. These two proteins are:

**3. Physiologic HA ICSI** 

and also has a number of other advantages:

development (Van den Bergh et al., 2010)

Barak et al., 2001; Van den Bergh et al., 2010)

hypothesized to have toxic effects on oocytes (Jean et al., 1996; 2001).

pipette (Balaban et al., 2003)

the heat shock protein HspA2 chaperone, involved in meiosis

and for this reason they are not able to fertilize the oocyte naturally.

creatine kinase (CK), abundant in the sperm cytoplasm (Cayli et al., 2003)

PICSI dishes are conventional plastic culture dishes pre-prepared with 3 microdots of powdered HA. The powdered HA is re-hydrated by adding a 5 µL droplets of fresh culture medium to each of the three microdots. A 2 µL droplet with suspension of treated spermatozoa is then connected with a pipette tip to these culture medium droplets. The PICSI dish is incubated under oil; within 5 minutes the bound spermatozoa are attached by their head to the surface of the HA-microdots and are spinning around their head (Figure 1).

Fig. 1. Spermatozoa in PICSI dish (magnification 400 X)

New Advances in Intracytoplasmic Sperm Injection (ICSI) 103

It has been demonstrated that the injection of HA-bound spermatozoa improves embryo quality and development by favouring selection of spermatozoa with normal nucleus and intact DNA: in fact, top-quality embryo rate is higher in HA–ICSI than in conventional PVP-ICSI and embryo development rate has also been found to be significantly increased (Parmegiani et al., 2010 a). Furthermore, HA-ICSI may speed up the time-consuming IMSI (Parmegiani et al., 2010 a). The largest study published to date as full article (428 patients) comparing physiologic HA-ICSI to conventional PVP-ICSI (Parmegiani et al., 2010 b) revealed that injection of HA-bound spermatozoa determines a statistically significant

A positive trend in fertilization and pregnancy rates - when injecting HA-bound spermatozoa – has been reported (Mènèzo & Nicollet, 2004). Nasr-Esfahani et al. (2008) have also published a study showing a higher fertilization rate when injecting oocytes with HA-

A statistically significant improvement in fertilization rate and embryo quality and a reduction in the number of miscarriages were found by Worrilow et al. (2007) performing PICSI versus conventional ICSI. In a subsequent study, the same authors demonstrated that PICSI significantly improves embryo quality, significantly reducing embryo fragmentation rate on day 3 and favours good blastocyst formation and clinical pregnancy

In contrast, one report found no differences in fertilization, pregnancy and implantation rates (Sanchez et al., 2005); this lack of significant clinical improvements after the injection of HA-bound spermatozoa may be due to the small number of patients studied (18). Recently, a historical comparison between 2014 HA-ICSI and 1920 PVP-ICSI showed no statistically significant increase in embryo quality and pregnancy rate for physiologic ICSI

Van den Berg et al., (2010) found no difference in zygote score when injecting, in a prospective randomized way, 407 sibling metaphase II oocytes, with either HA bound (HA+) or non-bound (HA-) spermatozoa. Our group (Parmegiani et al., 2010 c) questioned the ethical aspect of this study, which was based on the injection of HA non–bound

In conclusion, most of the studies cited above showed an improved clinical outcome of physiologic ICSI using HA-viscous medium or HA-dish (Parmegiani et al., 2010 a; 2010b; Worrilow et al., 2007; 2010; Nasr-Esfahani et al., 2008). At the very least, in all the studies physiologic ICSI never caused a detrimental effect on ICSI outcome parameters (Table 1). If larger multi-centre prospective-randomized studies confirm the suggested beneficial effects on ICSI outcome, HA should be considered the first choice for "physiologic" sperm selection prior to ICSI because of its capacity to reduce genetic complications and for its total lack of

FR: fertilization rate; EQ: embryo quality; PR: pregnancy rate; IR: implantation rate; MR:

spermatozoa, due to the risk of transmission of chromosomal anomalies.

**3.3 Clinical efficiency of "physiologic ICSI"** 

improvement in embryo quality and implantation.

selected spermatozoa.

rate (Worrilow et al., 2010).

(Mènèzo et al., 2010).

toxicity (Parmegiani et al., 2010 c).

miscarriage rate; ND: not described.

An ICSI injecting pipette is used to pick the best motile HA-bound sperm up and inject them one by one into an oocyte. The ICSI injecting pipette can be previously loaded with viscous medium (PVP or Sperm Slow) to facilitate sperm micromanipulation.

In PICSI, HA-sperm (\*) are bound by the head to the bottom of the dish and have vigorous motility with the tail spinning around their head. HA-unbound spermatozoa, in contrast, swim free all around the droplet of culture medium with varied motility.

## **3.2 Sperm slow procedure (***Parmegiani et al., 2010b***)**

On a culture dish (plastic or glass bottomed), a 2 µL droplet with suspension of treated spermatozoa is connected with a pipette tip to a 5 µL droplet of fresh culture medium. Simultaneously, a 5 µL droplet of Sperm Slow is connected with a pipette tip to the 5 µL droplet of fresh culture medium (Figure 2). The spermatozoa on this culture dish are incubated for 5 min at 37°C under oil. Spermatozoa bound to HA are slowed (as if trapped in a net) in the junction zone of the 2 droplets, these spermatozoa are selected and detached by injecting pipette and subsequently injected into oocytes. In Sperm Slow, HA-bound sperm tail appears stretched, its motility is dramatically slowed and its beats have narrow amplitude. HA non-bound spermatozoa swim all around the medium droplet, they are less slowed by the viscosity of the medium and their tail-beats have wider amplitude.

Fig. 2. Sperm Slow droplet preparation

A 2 µL droplet with suspension of treated spermatozoa is connected with a pipette tip to a 5 µL droplet of fresh culture medium. Simultaneously, a 5 µL droplet of Sperm Slow is connected with a pipette tip to the 5 µL droplet of fresh culture medium.

## **3.3 Clinical efficiency of "physiologic ICSI"**

102 Advances in Embryo Transfer

An ICSI injecting pipette is used to pick the best motile HA-bound sperm up and inject them one by one into an oocyte. The ICSI injecting pipette can be previously loaded with viscous

In PICSI, HA-sperm (\*) are bound by the head to the bottom of the dish and have vigorous motility with the tail spinning around their head. HA-unbound spermatozoa, in contrast,

On a culture dish (plastic or glass bottomed), a 2 µL droplet with suspension of treated spermatozoa is connected with a pipette tip to a 5 µL droplet of fresh culture medium. Simultaneously, a 5 µL droplet of Sperm Slow is connected with a pipette tip to the 5 µL droplet of fresh culture medium (Figure 2). The spermatozoa on this culture dish are incubated for 5 min at 37°C under oil. Spermatozoa bound to HA are slowed (as if trapped in a net) in the junction zone of the 2 droplets, these spermatozoa are selected and detached by injecting pipette and subsequently injected into oocytes. In Sperm Slow, HA-bound sperm tail appears stretched, its motility is dramatically slowed and its beats have narrow amplitude. HA non-bound spermatozoa swim all around the medium droplet, they are less

A 2 µL droplet with suspension of treated spermatozoa is connected with a pipette tip to a 5 µL droplet of fresh culture medium. Simultaneously, a 5 µL droplet of Sperm Slow is

connected with a pipette tip to the 5 µL droplet of fresh culture medium.

slowed by the viscosity of the medium and their tail-beats have wider amplitude.

medium (PVP or Sperm Slow) to facilitate sperm micromanipulation.

swim free all around the droplet of culture medium with varied motility.

**3.2 Sperm slow procedure (***Parmegiani et al., 2010b***)**

Fig. 2. Sperm Slow droplet preparation

It has been demonstrated that the injection of HA-bound spermatozoa improves embryo quality and development by favouring selection of spermatozoa with normal nucleus and intact DNA: in fact, top-quality embryo rate is higher in HA–ICSI than in conventional PVP-ICSI and embryo development rate has also been found to be significantly increased (Parmegiani et al., 2010 a). Furthermore, HA-ICSI may speed up the time-consuming IMSI (Parmegiani et al., 2010 a). The largest study published to date as full article (428 patients) comparing physiologic HA-ICSI to conventional PVP-ICSI (Parmegiani et al., 2010 b) revealed that injection of HA-bound spermatozoa determines a statistically significant improvement in embryo quality and implantation.

A positive trend in fertilization and pregnancy rates - when injecting HA-bound spermatozoa – has been reported (Mènèzo & Nicollet, 2004). Nasr-Esfahani et al. (2008) have also published a study showing a higher fertilization rate when injecting oocytes with HAselected spermatozoa.

A statistically significant improvement in fertilization rate and embryo quality and a reduction in the number of miscarriages were found by Worrilow et al. (2007) performing PICSI versus conventional ICSI. In a subsequent study, the same authors demonstrated that PICSI significantly improves embryo quality, significantly reducing embryo fragmentation rate on day 3 and favours good blastocyst formation and clinical pregnancy rate (Worrilow et al., 2010).

In contrast, one report found no differences in fertilization, pregnancy and implantation rates (Sanchez et al., 2005); this lack of significant clinical improvements after the injection of HA-bound spermatozoa may be due to the small number of patients studied (18). Recently, a historical comparison between 2014 HA-ICSI and 1920 PVP-ICSI showed no statistically significant increase in embryo quality and pregnancy rate for physiologic ICSI (Mènèzo et al., 2010).

Van den Berg et al., (2010) found no difference in zygote score when injecting, in a prospective randomized way, 407 sibling metaphase II oocytes, with either HA bound (HA+) or non-bound (HA-) spermatozoa. Our group (Parmegiani et al., 2010 c) questioned the ethical aspect of this study, which was based on the injection of HA non–bound spermatozoa, due to the risk of transmission of chromosomal anomalies.

In conclusion, most of the studies cited above showed an improved clinical outcome of physiologic ICSI using HA-viscous medium or HA-dish (Parmegiani et al., 2010 a; 2010b; Worrilow et al., 2007; 2010; Nasr-Esfahani et al., 2008). At the very least, in all the studies physiologic ICSI never caused a detrimental effect on ICSI outcome parameters (Table 1). If larger multi-centre prospective-randomized studies confirm the suggested beneficial effects on ICSI outcome, HA should be considered the first choice for "physiologic" sperm selection prior to ICSI because of its capacity to reduce genetic complications and for its total lack of toxicity (Parmegiani et al., 2010 c).

FR: fertilization rate; EQ: embryo quality; PR: pregnancy rate; IR: implantation rate; MR: miscarriage rate; ND: not described.

New Advances in Intracytoplasmic Sperm Injection (ICSI) 105

The conventional magnification for sperm evaluation at ICSI is a maximum 400 X. Some studies demonstrate that sperm morphology according to strict criteria (Kruger et al., 1986;

has little prognostic value in ICSI cycle outcomes (Svalander et al., 1996; French et al.,

But, it seem logical that the goal of obtaining the most viable embryo and reducing diseases in newborns is dependent on the selection of ideal gametes, both oocytes and spermatozoa (Parmegiani et al., 2010 c). Unfortunately, when observed at 400-1000 magnification, sperm dimension and shape are no reliable attributes for predicting chromatin integrity or presence of numerical chromosomal aberrations (Celik-Ozenci et al., 2004). To improve "imaging" sperm selection, the group of Bartoov (1994, 2001, 2002) developed a method of unstained, real-time, high magnification evaluation of spermatozoa (MSOME: motile sperm organelle morphology examination). MSOME is performed using an inverted light microscope equipped with high-power Nomarski optic enhanced by digital imaging to achieve a magnification of up to 6300 X (Figure 3). Application of MSOME selection in patients undergoing ICSI demonstrated that morphological integrity of the human sperm nucleus is an important parameter associated with pregnancy rate (Bartoov et al., 2003, Berkovitz et al., 2005). The modified ICSI procedure based on MSOME criteria was defined as IMSI: intracytoplasmic morphologically selected sperm injection (Bartoov et al., 2003). A matched study (Bartoov et al., 2003) revealed that pregnancy rate was significantly increased in IMSI as compared with routine ICSI, and implantation rate was even the 3-fold higher. Berkovitz et al., 2005 found an increase in abortion rate from 10% (no spermatozoa with normal nuclei) to 57% if no normal sperm for ICSI was available. In fact, ICSI outcome is significantly improved by the exclusive microinjection into the oocyte of spermatozoa with a strictly defined, morphologically normal nucleus, in couples with previous ICSI failures (Bartoov et al., 2003; Berkovitz et al., 2005; Hazout et al., 2006; Antinori et al., 2008; Franco et al., 2008; Mauri et al., 2010; Souza Setti et al., 2010) or with severe male factor (Balaban et al., 2011, Souza Setti et al., 2011). IMSI positive effect is not evident on day 2 embryos (Mauri et al., 2010) but, conversely, the injection of spermatozoa with abnormal sperm head or with nuclear vacuoles negatively affects embryo development in day 5-6 (Vanderzwalmen et al., 2008) and ICSI outcome (Berkovitz et al., 2006a; 2006b; Cassuto et al., 2009; Nadalini et al., 2009). The positive effect on ICSI outcome given by the injection may be due to the significantly better mitochondrial function, chromatin status and reduced aneuploidy rate of spermatozoa without nuclear vacuoles when compared with vacuolized spermatozoa (Garolla et al., 2008; Boitrelle, et al., 2011). In addition, spermatozoa free of nuclear morphological malformations are related with lower incidence of aneuploidy in

It should be mentioned that IMSI is a time-consuming procedure: selecting a "normal" MSOME spermatozoon requires 60-120 minutes (Antinori et al., 2008). Furthermore, the process of searching for spermatozoa at high magnification may itself damage sperm cytoplasm: sperm nucleus vacuolization significantly increases after 2 hours on the microscope's heated stage (Peer et al., 2007). IMSI procedure can be speeded up by merging

**5. Intracytoplasmic morphologically selected sperm injection (IMSI)** 

does not influence embryo development or morphology (French et al., 2010)

1988):

2010)

derived embryos (Figueira et al., 2011).


Table 1. Studies on injection of HA-bound spermatozoa

## **4. Zona - Bound spermatozoa**

Immature spermatozoa have a low density of ZP binding sites as well as HA receptors (Huszar et al., 2003). Human sperm bound to ZP exhibit attributes similar to those of HAbound sperm, including minimal DNA fragmentation, normal shape, and low frequency of chromosomal aneuploidies (Yagcy et al., 2010). Furthermore, in some mammals, the same sperm membrane protein is involved firstly in hyaluronidase activity and subsequently in ZP binding (Hunnicutt et al., 1996). These findings suggest that the spermatozoa–ZP binding process plays an important role in the natural selection of spermatozoa as well as HA.

A spermatozoa-ZP binding test can be performed by culturing spermatozoa for a couple of hours with immature metaphase I oocytes; the spermatozoa bound to ZP can be recovered with an injecting pipette and used for ICSI: when using this system Paes de Almeida Ferreira Braga et al. (2009) found that the injection of ZP-bound spermatozoa increases embryo quality. Black et al. (2010) observed a trend in implantation and clinical pregnancy rates when injecting ZP-bound spermatozoa in a study on ZP-ICSI versus conventional ICSI. Liu et al. (2011) observed a significant improvement in top embryo quality rate comparing ZP-ICSI with conventional ICSI.

Even though at the present time there is little information regarding all the factors involved in sperm-ZP binding and its mechanism, these last studies suggest that the spermatozoa–ZP binding test may be an efficient method for identifying competent spermatozoa for ICSI. ZP selection could then be coupled to HA selection in order to replicate the natural path of the spermatozoa towards the oocyte.

Worrilow et al, 2007 PICSI 240 couples: PICSI vs PVP-ICSI Improvement in FR, EQ, MR

50 couples: sibling oocytes; HA-

44 couples: sibling oocytes; HA+

Worrilow et al, 2010 PICSI 215 couples: PICSI vs PVP-ICSI Improvement in EQ

Sanchez et al, 2005 N.D. 18 HA-ICSI versus control group No differences

*System N° of treatments or patients HA-ICSI determines :* 

ICSI vs PVP-ICSI Improvement FR

vs HA- sperms No differences

Slow 331 HA-ICSI vs 97 PVP-ICSI Improvement in EQ, IR

Slow 125 HA-ICSI vs 107 PVP-ICSI Improvement in EQ

Slow 2014 HA-ICSI vs 1920 PVP-ICSI No differences

Immature spermatozoa have a low density of ZP binding sites as well as HA receptors (Huszar et al., 2003). Human sperm bound to ZP exhibit attributes similar to those of HAbound sperm, including minimal DNA fragmentation, normal shape, and low frequency of chromosomal aneuploidies (Yagcy et al., 2010). Furthermore, in some mammals, the same sperm membrane protein is involved firstly in hyaluronidase activity and subsequently in ZP binding (Hunnicutt et al., 1996). These findings suggest that the spermatozoa–ZP binding process plays an important role in the natural selection of

A spermatozoa-ZP binding test can be performed by culturing spermatozoa for a couple of hours with immature metaphase I oocytes; the spermatozoa bound to ZP can be recovered with an injecting pipette and used for ICSI: when using this system Paes de Almeida Ferreira Braga et al. (2009) found that the injection of ZP-bound spermatozoa increases embryo quality. Black et al. (2010) observed a trend in implantation and clinical pregnancy rates when injecting ZP-bound spermatozoa in a study on ZP-ICSI versus conventional ICSI. Liu et al. (2011) observed a significant improvement in top embryo quality rate comparing

Even though at the present time there is little information regarding all the factors involved in sperm-ZP binding and its mechanism, these last studies suggest that the spermatozoa–ZP binding test may be an efficient method for identifying competent spermatozoa for ICSI. ZP selection could then be coupled to HA selection in order to replicate the natural path of the

Slow 92 HA-ICSI vs 110 PVP-ICSI No differences

*Authors HA-*

Sperm

homemade

Sperm Slow

Sperm

Sperm

Table 1. Studies on injection of HA-bound spermatozoa

Menezo et Nicollet,

Nasr-Esfahani et al, 2008

Van Den Berg et al, 2009

2010 a

2010 b

Parmegiani et al,

Parmegiani et al,

Menezo et al, 2010 Sperm

spermatozoa as well as HA.

ZP-ICSI with conventional ICSI.

spermatozoa towards the oocyte.

**4. Zona - Bound spermatozoa** 

2004

## **5. Intracytoplasmic morphologically selected sperm injection (IMSI)**

The conventional magnification for sperm evaluation at ICSI is a maximum 400 X. Some studies demonstrate that sperm morphology according to strict criteria (Kruger et al., 1986; 1988):


But, it seem logical that the goal of obtaining the most viable embryo and reducing diseases in newborns is dependent on the selection of ideal gametes, both oocytes and spermatozoa (Parmegiani et al., 2010 c). Unfortunately, when observed at 400-1000 magnification, sperm dimension and shape are no reliable attributes for predicting chromatin integrity or presence of numerical chromosomal aberrations (Celik-Ozenci et al., 2004). To improve "imaging" sperm selection, the group of Bartoov (1994, 2001, 2002) developed a method of unstained, real-time, high magnification evaluation of spermatozoa (MSOME: motile sperm organelle morphology examination). MSOME is performed using an inverted light microscope equipped with high-power Nomarski optic enhanced by digital imaging to achieve a magnification of up to 6300 X (Figure 3). Application of MSOME selection in patients undergoing ICSI demonstrated that morphological integrity of the human sperm nucleus is an important parameter associated with pregnancy rate (Bartoov et al., 2003, Berkovitz et al., 2005). The modified ICSI procedure based on MSOME criteria was defined as IMSI: intracytoplasmic morphologically selected sperm injection (Bartoov et al., 2003). A matched study (Bartoov et al., 2003) revealed that pregnancy rate was significantly increased in IMSI as compared with routine ICSI, and implantation rate was even the 3-fold higher. Berkovitz et al., 2005 found an increase in abortion rate from 10% (no spermatozoa with normal nuclei) to 57% if no normal sperm for ICSI was available. In fact, ICSI outcome is significantly improved by the exclusive microinjection into the oocyte of spermatozoa with a strictly defined, morphologically normal nucleus, in couples with previous ICSI failures (Bartoov et al., 2003; Berkovitz et al., 2005; Hazout et al., 2006; Antinori et al., 2008; Franco et al., 2008; Mauri et al., 2010; Souza Setti et al., 2010) or with severe male factor (Balaban et al., 2011, Souza Setti et al., 2011). IMSI positive effect is not evident on day 2 embryos (Mauri et al., 2010) but, conversely, the injection of spermatozoa with abnormal sperm head or with nuclear vacuoles negatively affects embryo development in day 5-6 (Vanderzwalmen et al., 2008) and ICSI outcome (Berkovitz et al., 2006a; 2006b; Cassuto et al., 2009; Nadalini et al., 2009). The positive effect on ICSI outcome given by the injection may be due to the significantly better mitochondrial function, chromatin status and reduced aneuploidy rate of spermatozoa without nuclear vacuoles when compared with vacuolized spermatozoa (Garolla et al., 2008; Boitrelle, et al., 2011). In addition, spermatozoa free of nuclear morphological malformations are related with lower incidence of aneuploidy in derived embryos (Figueira et al., 2011).

It should be mentioned that IMSI is a time-consuming procedure: selecting a "normal" MSOME spermatozoon requires 60-120 minutes (Antinori et al., 2008). Furthermore, the process of searching for spermatozoa at high magnification may itself damage sperm cytoplasm: sperm nucleus vacuolization significantly increases after 2 hours on the microscope's heated stage (Peer et al., 2007). IMSI procedure can be speeded up by merging

New Advances in Intracytoplasmic Sperm Injection (ICSI) 107

normal shape and normal chromatin content. For rapid evaluation of nuclear normalcy, a fixed, transparent, celluloid form of a sperm nucleus fitting MSOME criteria for length and widths can be superimposed on the examined cell on the screen: the nuclear shape is considered abnormal if it differs in length or width by 2 standard deviations from the normal mean axes values; vacuoles can be examined using a similar celluloid form (Figure 4). Alternatively, spermatozoa can be measured for nuclear length, width and vacuoles

Fig. 4. IMSI procedure. Human Spermatozoa (magnification >10'000 X)

similar celluloid form (Bartoov et al., 2003).

**6. Sperm head birefringence** 

For evaluation of nuclear normalcy, a fixed, transparent, celluloid form of a sperm nucleus fitting MSOME criteria for length and widths is superimposed on the examined cell on the screen: the nuclear shape is considered abnormal if it differs in length or width by 2 standard deviations from the normal mean axes values; vacuoles are examined using a

A new tool for sperm selection is the application of polarization microscopy to ICSI (Baccetti, 2004). This method is based on the birefringence characteristics of the sperm protoplasmic texture. In the mature sperm nucleus, there is a strong intrinsic birefringence associated with nucleoprotein filaments that are ordered in rods and longitudinally oriented. An inverted microscope specifically equipped with polarizing lenses allows for the real-time selection of birefringent spermatozoa for ICSI. The localization of the birefringence in the postacrosomial region indicates that the acrosomial reaction has already occurred; the

with specific digital imaging softwares

of high magnification microscopy together with HA-sperm selection. In fact, a HA-medium may help to select a sub-population of spermatozoa with normal nucleus according to MSOME criteria: Parmegiani et al (2010a) found that nucleus normalcy rate was significantly higher in HA-bound spermatozoa than in spermatozoa in PVP.

It can be concluded that, despite the time consuming procedure and the cost of the instrument for high magnification microscopy, IMSI has proved itself a valid tool for safe, non-invasive sperm selection and it can be widely applied in the near future.

Fig. 3. Human Spermatozoa (magnification >6300 X)

#### **5.1 IMSI procedure**

Spermatozoa are generally first treated with a density gradient system. Then, the prepared sperm suspension is put into a PVP (or Sperm Slow in case of "Physiologic IMSI", *Parmegiani et al., 2010 a*) droplet, on a glass-bottom culture dish under oil. In order to choose the best spermatozoa to inject, sperm "nucleus normalcy" is evaluated. The nucleus normalcy is assessed in real time according to Motile Sperm Organelle Morphology Examination (MSOME) criteria. According to MSOME criteria, normallyshaped sperm nucleus is smooth, symmetric, and with oval configuration. Average lengths and widths (± Standard Deviation) must be 4.75±0.28 µm and 3.28 ±0.20 µm, respectively. Nuclear chromatin content is considered abnormal if sperm head contains one or more vacuoles (diameter of 0.78±0.18) that occupy more than 4% of the normal nuclear area. To be considered morphologically normal, a sperm nucleus has to have both

of high magnification microscopy together with HA-sperm selection. In fact, a HA-medium may help to select a sub-population of spermatozoa with normal nucleus according to MSOME criteria: Parmegiani et al (2010a) found that nucleus normalcy rate was

It can be concluded that, despite the time consuming procedure and the cost of the instrument for high magnification microscopy, IMSI has proved itself a valid tool for safe,

Spermatozoa are generally first treated with a density gradient system. Then, the prepared sperm suspension is put into a PVP (or Sperm Slow in case of "Physiologic IMSI", *Parmegiani et al., 2010 a*) droplet, on a glass-bottom culture dish under oil. In order to choose the best spermatozoa to inject, sperm "nucleus normalcy" is evaluated. The nucleus normalcy is assessed in real time according to Motile Sperm Organelle Morphology Examination (MSOME) criteria. According to MSOME criteria, normallyshaped sperm nucleus is smooth, symmetric, and with oval configuration. Average lengths and widths (± Standard Deviation) must be 4.75±0.28 µm and 3.28 ±0.20 µm, respectively. Nuclear chromatin content is considered abnormal if sperm head contains one or more vacuoles (diameter of 0.78±0.18) that occupy more than 4% of the normal nuclear area. To be considered morphologically normal, a sperm nucleus has to have both

significantly higher in HA-bound spermatozoa than in spermatozoa in PVP.

non-invasive sperm selection and it can be widely applied in the near future.

Fig. 3. Human Spermatozoa (magnification >6300 X)

**5.1 IMSI procedure** 

normal shape and normal chromatin content. For rapid evaluation of nuclear normalcy, a fixed, transparent, celluloid form of a sperm nucleus fitting MSOME criteria for length and widths can be superimposed on the examined cell on the screen: the nuclear shape is considered abnormal if it differs in length or width by 2 standard deviations from the normal mean axes values; vacuoles can be examined using a similar celluloid form (Figure 4). Alternatively, spermatozoa can be measured for nuclear length, width and vacuoles with specific digital imaging softwares

Fig. 4. IMSI procedure. Human Spermatozoa (magnification >10'000 X)

For evaluation of nuclear normalcy, a fixed, transparent, celluloid form of a sperm nucleus fitting MSOME criteria for length and widths is superimposed on the examined cell on the screen: the nuclear shape is considered abnormal if it differs in length or width by 2 standard deviations from the normal mean axes values; vacuoles are examined using a similar celluloid form (Bartoov et al., 2003).

## **6. Sperm head birefringence**

A new tool for sperm selection is the application of polarization microscopy to ICSI (Baccetti, 2004). This method is based on the birefringence characteristics of the sperm protoplasmic texture. In the mature sperm nucleus, there is a strong intrinsic birefringence associated with nucleoprotein filaments that are ordered in rods and longitudinally oriented. An inverted microscope specifically equipped with polarizing lenses allows for the real-time selection of birefringent spermatozoa for ICSI. The localization of the birefringence in the postacrosomial region indicates that the acrosomial reaction has already occurred; the

New Advances in Intracytoplasmic Sperm Injection (ICSI) 109

Furthermore, some of these new technologies also help the standardization of ICSI, reducing intra-operator and inter-operator variability in choosing the spermatozoon to inject. For example, HA-ICSI offers to the embryologist the possibility to recognize the spermatozoa which have completed the maturation process. On the other hand, IMSI allows a precise sperm evaluation and measurement. In particular, these two techniques may also be merged together, pre-selecting HA-bound spermatozoa before High –magnification evaluation. This combined procedure (Physiologic IMSI) speeds up the "time consuming" sperm selection

The easy reproducibility of these new advances in ICSI should encourage the embryologists and clinicians to automatically offer these technical improvements to all ICSI patients, not only to optimize clinical results but most of all to restore some basic check-points of natural

Ahmad L, Jalali S, Shami SA, Akram Z. (2007) Sperm preparation: DNA damage by comet

Antinori M, Licata E, Dani G, Cerusico F, Versaci C, D'angelo D, Antinori S. (2008)

Baccetti B. (2004) Microscopical advances in assisted reproduction. *J Submicrosc Cytol Pathol*

Balaban B, Lundin K, Morrell JM, Tjellström H, Urman B, Holmes PV. (2003) An alternative

Balaban B, Yakin K, Alatas C, Oktem O, Isiklar A, Urman B. (2011) Clinical outcome of

Bartoov B, Berkovitz A, Eltes F. (2001) Selection of spermatozoa with normal nuclei to

Intracytoplasmic morphologically selected sperm injection: a prospective

intracytoplasmic injection of spermatozoa morphologically selected under high magnification: a prospective randomized study. *Reprod Biomed Online* 22, 472-476 Barak Y, Menezo Y, Veiga A, Elder K. (2001) A physiological replacement for polyvinylpyrrolidone (PVP) in assisted reproductive technology. *Hum Fert* 4, 99-103. Bartoov B, Eltes F, Pansky M, Langzam J, Reichart M, Soffer Y. (1994) Improved diagnosis of

male fertility potential via a combination of quantitative ultramorphology and

improve the pregnancy rate with intracytoplasmic sperm injection. *N Engl J Med*

assay in normo- and teratozoospermics. *Arch. Androl* 53, 325-338.

to PVP for slowing sperm prior to ICSI. *Hum Reprod* 18, 1887-1889.

 fertilization embryo quality blastocyst formation

pregnancy

reduction in abortion.

**8. Acknowledgment** 

36, 333-339.

345, 1067-1068.

**9. References** 

according to MSOME criteria (Parmegiani et al, 2010 a)

fertilization which are bypassed in the conventional ICSI.

The authors wish to thank Ms Maggie Baigent for revising the manuscript.

randomized trial. *Reprod Biomed Online* 16, 835-841.

routine semen analyses. *Hum Reprod* 9, 2069-2075.

injection of acrosome reacted spermatozoa seems to favour the development of viable ICSI embryos (Gianaroli et al., 2010). The injection of birefringent spermatozoa seems to be useful, especially in cases of oligoasthenoteratozoospermia or testicular spermatozoa (Gianaroli et al., 2008; 2010).

## **7. Conclusions**

The introduction of ICSI (Palermo et al., 1992) has changed in a revolutionary way the world of assisted reproduction technology allowing us to efficiently treat patients with:


In these situations, suboptimal spermatozoa could by-pass the physiological check-points of natural fertilization and generate embryos, and subsequently babies. Conventional ICSI has the hypothetical risk of injecting immature, DNA damaged, aneuploid, low motile, morphologically abnormal, zona binding deficient, poor acrosome reacted, spermatozoa. Nowadays, we have no real knowledge of the effects of suboptimal sperm selection on ICSI adults in the long term, at least for humans. A potentially worrying aspect of the injection of DNA damaged spermatozoa for example, has been suggested by studies performed on animals which showed not only a negative effect on pregnancy and birth, but also later side effects on the health of adult animals such as aberrant growth, premature ageing, abnormal behaviour, and mesenchymal tumours (Fernandez-Gonzales et al., 2008).

Fortunately, in humans, the risk of injecting DNA damaged spermatozoa seems to be minimized by classical sperm preparation techniques prior to ICSI (Zini et al. 2000; Younglai et al., 2001; Donnelly et al., 2000; Ahmad et al., 2007; Jackson et al., 2010; Marchesi et al., 2010; Castillo et al., 2011: Ebner et al, 2011) and follow-up studies on ICSI children have demonstrated the safety of this technique (Van Steirteghem et al., 2002, Leunens et al., 2008; Belva et al., 2011; Woldringh et al., 2011) although a slight increase of chromosome aberration seems to be caused by the injection of aneuploid spermatozoa (Bonduelle et al., 2002).

The recent refinements of the ICSI procedure described in this chapter, are reliable, easy-todo, non-invasive and in some cases "closest to the nature" than the conventional procedure. For example, selecting spermatozoa prior to ICSI by their maturation markers such as HA-ZP receptors (Huszar et al., 2003; Paes de Almeida Ferreira Braga et al., 2009) it is possible at very least to mimic nature in order to restore physiological selection and prevent hypothetical fertilization by DNA damaged and chromosomal unbalanced spermatozoa. In addition, non-invasive imaging sperm selection techniques such as IMSI (Bartoov et al., 2003) or sperm head birefringence (Gianaroli et al., 2008) can be valid tools for helping in selection of the ideal spermatozoa.

In fact, sperm selection based on non invasive morphology or maturity markers helps the embryologist in selection of the "ideal" spermatozoa to inject. These new advances in ICSI may allow the selection of the spermatozoa contributing to the improve:

fertilization

108 Advances in Embryo Transfer

injection of acrosome reacted spermatozoa seems to favour the development of viable ICSI embryos (Gianaroli et al., 2010). The injection of birefringent spermatozoa seems to be useful, especially in cases of oligoasthenoteratozoospermia or testicular spermatozoa

The introduction of ICSI (Palermo et al., 1992) has changed in a revolutionary way the world

In these situations, suboptimal spermatozoa could by-pass the physiological check-points of natural fertilization and generate embryos, and subsequently babies. Conventional ICSI has the hypothetical risk of injecting immature, DNA damaged, aneuploid, low motile, morphologically abnormal, zona binding deficient, poor acrosome reacted, spermatozoa. Nowadays, we have no real knowledge of the effects of suboptimal sperm selection on ICSI adults in the long term, at least for humans. A potentially worrying aspect of the injection of DNA damaged spermatozoa for example, has been suggested by studies performed on animals which showed not only a negative effect on pregnancy and birth, but also later side effects on the health of adult animals such as aberrant growth, premature ageing, abnormal

Fortunately, in humans, the risk of injecting DNA damaged spermatozoa seems to be minimized by classical sperm preparation techniques prior to ICSI (Zini et al. 2000; Younglai et al., 2001; Donnelly et al., 2000; Ahmad et al., 2007; Jackson et al., 2010; Marchesi et al., 2010; Castillo et al., 2011: Ebner et al, 2011) and follow-up studies on ICSI children have demonstrated the safety of this technique (Van Steirteghem et al., 2002, Leunens et al., 2008; Belva et al., 2011; Woldringh et al., 2011) although a slight increase of chromosome aberration seems to be caused by the injection of aneuploid spermatozoa

The recent refinements of the ICSI procedure described in this chapter, are reliable, easy-todo, non-invasive and in some cases "closest to the nature" than the conventional procedure. For example, selecting spermatozoa prior to ICSI by their maturation markers such as HA-ZP receptors (Huszar et al., 2003; Paes de Almeida Ferreira Braga et al., 2009) it is possible at very least to mimic nature in order to restore physiological selection and prevent hypothetical fertilization by DNA damaged and chromosomal unbalanced spermatozoa. In addition, non-invasive imaging sperm selection techniques such as IMSI (Bartoov et al., 2003) or sperm head birefringence (Gianaroli et al., 2008) can be valid tools for helping in

In fact, sperm selection based on non invasive morphology or maturity markers helps the embryologist in selection of the "ideal" spermatozoa to inject. These new advances in ICSI

may allow the selection of the spermatozoa contributing to the improve:

of assisted reproduction technology allowing us to efficiently treat patients with:

behaviour, and mesenchymal tumours (Fernandez-Gonzales et al., 2008).

(Gianaroli et al., 2008; 2010).

oligoasthenoteratozoospermia

 testicular spermatozoa limited number of oocytes previous IVF failures

(Bonduelle et al., 2002).

selection of the ideal spermatozoa.

**7. Conclusions** 


Furthermore, some of these new technologies also help the standardization of ICSI, reducing intra-operator and inter-operator variability in choosing the spermatozoon to inject. For example, HA-ICSI offers to the embryologist the possibility to recognize the spermatozoa which have completed the maturation process. On the other hand, IMSI allows a precise sperm evaluation and measurement. In particular, these two techniques may also be merged together, pre-selecting HA-bound spermatozoa before High –magnification evaluation. This combined procedure (Physiologic IMSI) speeds up the "time consuming" sperm selection according to MSOME criteria (Parmegiani et al, 2010 a)

The easy reproducibility of these new advances in ICSI should encourage the embryologists and clinicians to automatically offer these technical improvements to all ICSI patients, not only to optimize clinical results but most of all to restore some basic check-points of natural fertilization which are bypassed in the conventional ICSI.

## **8. Acknowledgment**

The authors wish to thank Ms Maggie Baigent for revising the manuscript.

## **9. References**


New Advances in Intracytoplasmic Sperm Injection (ICSI) 111

Dozortsev D, Rybouchkin A, De Sutter P, Dhont M. (1995) Sperm plasma membrane

Ebner T, Shebl O, Moser M, Mayer RB, Arzt W, Tews G. (2011) Easy sperm processing

Fernandez-Gonzalez R, Moreira PN, Perez-Crespo M, Sanchez-Martin M, Ramirez MA,

Figueira R de C, Braga DP, Setti AS, Iaconelli A Jr, Borges E Jr. (2011). Morphological nuclear

Franco JG Jr, Baruffi RL, Mauri AL, Petersen CG, Oliveira JB, Vagnini L. (2008) Significance

French DB, Sabanegh ES Jr, Goldfarb J, Desai N. (2010) Does severe teratozoospermia affect

Garolla A, Fortini D, Menegazzo M, De Toni L, Nicoletti V, Moretti A, Selice R, Engl B,

Gianaroli L, Magli MC, Collodel G, Moretti E, Ferraretti AP, Baccetti B. (2008) Sperm head's birefringence: a new criterion for sperm selection. *Fertil Steril* 90, 104-112. Gianaroli L, Magli MC, Ferraretti AP, Crippa A, Lappi M, Capitani S, Baccetti B. (2010)

spermatozoa for intracytoplasmic sperm injection. *Fertil Steril* 93, 807-813. Hazout A, Dumont-Hassan M, Junca AM, Cohen BP, Tesarik J. (2006) High-magnification

Heytens E, Parrington J, Coward K, Young C, Lambrecht S, Yoon SY, Fissore RA, Hamer R,

Hunnicutt GR, Primakoff P, Myles DG. (1996) Sperm surface protein PH-20 is bifunctional:

Huszar G & Vigue L. (1993) Incomplete development of human spermatozoa is associated

Huszar G, Stone K, Dix D, Vigue L. (2000) Putative creatine kinase M-isoform in human

nucleus decondensation. *Hum Reprod* 10, 2960-2964.

free spermatozoa. *Reprod Biomed Online* 22, 37-43.

screening cycle outcomes. *Fertil Steril* 95, 990-993.

physiological status. *Reprod Biomed Online* 17, 610-616.

secondary sperm-zona binding. *Biol Reprod* 55, 80-86.

morphology. *Mol Reprod Dev* 34, 292-298.

761-772.

12, 19-25.

2417-2428.

925-932.

*Biomed Online* 17, 42-45.

cycles? *Fertil Steril* 93, 1097-1103.

damage prior to intracytoplasmic sperm injection: a necessary condition for sperm

technique allowing exclusive accumulation and later usage of DNA-strandbreak-

Pericuesta E, Bilbao A, Bermejo-Alvarez P, de Dios HJ, De Fonseca FR, Gutiérrez-Adán A. (2008) Long-term effects of mouse intracytoplasmic sperm injection with DNA-fragmented sperm on health and behavior of adult offspring. *Biol Reprod* 78,

integrity of sperm cells is associated with preimplantation genetic aneuploidy

of large nuclear vacuoles in human spermatozoa: implications for ICSI. *Reprod* 

blastocyst formation, live birth rate, and other clinical outcome parameters in ICSI

Foresta C. (2008) High-power microscopy for selecting spermatozoa for ICSI by

Birefringence characteristics in sperm heads allow for the selection of reacted

ICSI overcomes paternal effect resistant to conventional ICSI. *Reprod Biomed Online*

Deane CM, Ruas M, Grasa P, Soleimani R, Cuvelier CA, Gerris J, Dhont M, Deforce D, Leybaert L, De Sutter P. (2009) Reduced amounts and abnormal forms of phospholipase C zeta (PLCζ) in spermatozoa from infertile men. *Hum Reprod* 24,

one activity is a hyaluronidase and a second, distinct activity is required in

with increased creatine phosphokinase concentration and abnormal head

sperm is identified as the 70-kilodalton heat shock protein HspA2. *Biol Reprod* 63,


Bartoov B, Berkovitz A, Eltes F, Kogosowski A, Menezo Y, Barak Y. (2002) Real-time fine

Bartoov B, Berkovitz A, Eltes F, Kogosovsky A, Yagoda A, Lederman H, Artzi S, Gross M,

Belva F, De Schrijver F, Tournaye H, Liebaers I, Devroey P, Haentjens P, Bonduelle M. (2011)

Berkovitz A, Eltes F, Yaari S, Katz N, Barr I, Fishman A, Bartoov B. (2005) The

Berkovitz A, Eltes F, Ellenbogen A, Peer S, Feldberg D, Bartoov B. (2006a) Does the presence

Berkovitz A, Eltes F, Lederman H, Peer S, Ellenbogen A, Feldberg B, Bartoov B. (2006b) How

Black M, Liu de Y, Bourne H, Baker HW. (2010) Comparison of outcomes of conventional

bound to the zona pellucida of immature oocytes. *Fertil Steril* 93, 672-674. Boitrelle F, Ferfouri F, Petit JM, Segretain D, Tourain C, Bergere M, Bailly M, Vialard F.

Bonduelle M, Van Assche.E, Joris H, Keymolen K, Devroey P, Van Steriteghem A, Liebaers I.

Cassuto NG, Bouret D, Plouchart JM, Jellad S, Vanderzwalmen P, Balet R, Larue L, Barak Y.

Celik-Ozenci C, Jakab A, Kovacs T, Catalanotti J, Demir R, Bray-Ward P, Ward D, Huszar G.

presence of numerical chromosomal aberrations. *Hum Reprod* 19, 2052-2059. Donnelly ET, O'Connell M, McClure N, Lewis SE. (2000) Differences in nuclear DNA

for fertilization and improved embryo quality. *Fertil Steril* 92, 1616-1625. Cayli S, Jakab A, Ovari L, Delpiano E, Celik-Ozenci C, Sakkas D, Ward D, Huszar G. (2003)

chromatin condensation. *Hum Reprod* 7,1650-1658.

*Androl* 23, 1-8.

*Steril* 80, 1413-1419

*Reprod* 7, 1752-1758.

*Hum Reprod* 21, 1787-1790.

J Androl 32, 324-32.

*Reprod Biomed Online* 7, 462-468.

spermatozoa. *Hum Reprod* 15, 1552-1561.

185-190.

morphology of motile human sperm cells is associated with IVF-ICSI outcome. *J* 

Barak Y. (2003) Pregnancy rates are higher with intracytoplasmic morphologically selected sperm injection than with conventional intracytoplasmic injection. *Fertil* 

Neonatal outcome of 724 children born after ICSI using non-ejaculated sperm. *Hum* 

morphological normalcy of the sperm nucleus and pregnancy rate of intracytoplasmic injection with morphologically selected sperm. *Hum Reprod* 20,

of nuclear vacuoles in human sperm selected for ICSI affect pregnancy outcome?

to improve IVF-ICSI outcome by sperm selection. *Reprod Biomed Online* 12, 634-638.

intracytoplasmic sperm injection and intracytoplasmic sperm injection using sperm

Albert M. Selva J. (2011) Large human sperm vacuoles observed in motile spermatozoa under high magnification: nuclear thumbprints linked to failure of

(2002) Prenatal testing in ICSI pregnancies: incidence of chromosomal anomalies in 1586 karyotypes and relation to sperm parameters. *Hum Reprod* 17, 2600-2614. Castillo J, Simon L, de Mateo S, Lewis S, Oliva R.(2011) Protamine/DNA ratios and DNA

damage in native and density gradient centrifugated sperm from infertile patients.

(2009) A new real-time morphology classification for human spermatozoa: a link

Biochemical markers of sperm function: male fertility and sperm selection for ICSI.

(2004) Sperm selection for ICSI: shape properties do not predict the absence or

fragmentation and mitochondrial integrity of semen and prepared human


New Advances in Intracytoplasmic Sperm Injection (ICSI) 113

Navarro-Costa P, Nogueira P, Carvalho M, Leal F, Cordeiro I, Calhaz-Jorge C, Gonçalves J,

Nadalini M, Tarozzi N, Distratis V, Scaravelli G, Borini A (2009) Impact of intracytoplasmic

Nasr-Esfahani MH, Razavi S, Vahdati AA, Fathi F, Tavalaee M (2008) Evaluation of sperm

Oehninger S. (2011) Clinical management of male infertility in assisted reproduction: ICSI

Paes Almeida Ferreira de Braga D, Iaconelli A Jr, Cassia Savio de FR, Madaschi C, Semiao-

Palermo G, Joris H, Devroey P, Van Steirteghem AC. (1992) Pregnancies after

Parmegiani L, Cognigni GE, Bernardi S, Troilo E, Ciampaglia W, Filicori M (2010a)

Parmegiani L, Cognigni GE, Ciampaglia W, Pocognoli P, Marchi F, Filicori M. (2010b) Efficiency of hyaluronic acid (HA) sperm selection. *J Assist Reprod Genet* 27, 13-16. Parmegiani L, Cognigni GE, Filicori M. (2010c) Risks in injecting hyaluronic acid non-bound

Peer S, Eltes F, Berkovitz A, Yehuda R, Itsykson P, Bartoov B. (2007) Is fine morphology of

Prinosilova P, Kruger T, Sati L, Ozkavukcu S, Vigue L, Kovanci E, Huszar G. (2009)

Sakkas D, Bianchi PG, Manicardi GC. (1997) Chromatin packaging anomalies and DNA

Sanchez M, Aran B, Blanco J, Vidal F, Veiga A, Barri PN, Huszar G. (2005) Preliminary

Schatten H & Sun QY. (2007) The role of centrosomes in mammalian fertilization and its

Souza Setti A, Ferreira RC, Paes de Almeida Ferreira Braga D, de Cássia Sávio Figueira R,

the human sperm nuclei affected by in vitro incubation at 37 degrees C? *Fertil Steril*

Selectivity of hyaluronic acid binding for spermatozoa with normal Tygerberg

damage in human sperm: their possible implications in the treatment of male factor infertility. In: *Genetics of human male infertility* (eds Barratt C, De Jonge C, Mortimer

clinical and FISH results on hyaluronic acid sperm selection to improve ICSI. *Hum* 

Iaconelli A Jr, Borges E J. (2010) Intracytoplasmic sperm injection outcome versus intracytoplasmic morphologically selected sperm injection outcome: a meta-

associates with defective human sperm. *Hum Reprod* 25, 2647-2654.

review. *Reprod Biomed Online* 19 (Supp l3), 45-55

*Assist Reprod Genet* 25, 197-203.

quality. *Fertil Steril* 93, 598-604.

spermatozoa. *Reprod Biomed Online* 20,437-438.

strict morphology. *Reprod Biomed Online* 18, 177-183.

D, Parinaud J), pp 205-221. Editions EDK, Paris, UK.

significance for ICSI. *Mol Hum Reprod* 15, 531-538.

analysis. *Reprod Biomed Online* 21, 450-455

802-807.

340(8810),17-18.

88, 1589-1594.

*Reprod* 20 (Supp1), i200.

and beyond. Int J Androl, 34:e319-329

Plancha CE (2010) Incorrect DNA methylation of the DAZL promoter CpG island

morphologically selected sperm injection on assisted reproduction outcome: a

selection procedure based on hyaluronic acid binding ability on ICSI outcome. *J* 

Francisco L, Borges E Jr. (2009) Outcome of ICSI using zona pellucida-bound spermatozoa and conventionally selected spermatozoa. *Reprod Biomed Online* 19,

intracytoplasmic injection of single spermatozoon into an oocyte. *Lancet* 

"Physiologic ICSI": hyaluronic acid (HA) favors selection of spermatozoa without DNA fragmentation and with normal nucleus, resulting in improvement of embryo


Huszar G, Ozenci CC, Cayli S, Zavaczki Z, Hansch E, Vigue L. (2003) Hyaluronic acid

Huszar G, Jakab A, Sakkas D, Ozenci CC, Cayli S, Delpiano E, Ozkavukcu S. (2007). Fertility

Jackson RE, Bormann CL, Hassun PA, Rocha AM, Motta EL, Serafini PC, Smith GD.(2010)

Jakab A, Sakkas D, Delpiano E, Cayli S, Kovanci E, Ward D, Revelli A, Huszar G. (2005)

Jean M, Barriere P and Mirallie S. (1996) Intracytoplasmic sperm injection without polyvinylpyrrolidone: an essential precaution? *Hum Reprod* 11,2332. Jean M, Mirallie S, Boudineau M, Tatin C and Barriere P. (2001) Intracytoplasmic sperm injection with polyvinylpyrrolidone: a potential risk. Fertil Steril, 76,419-420. Kruger TF, Menkveld R, Stander FS, Lombard CJ, Van der Merwe JP, van Zyl JA, Smith K.

Kruger TF, Acosta AA, Simmons KF, Swanson RJ, Matta JF, Oehninger S. (1988) Predictive

Leunens L, Celestin-Westreich S, Bonduelle M, Liebaers I, Ponjaert-Kristoffersen I. (2008)

Liu F, Qiu Y, Zou Y, Deng ZH, Yang H, Liu DY. (2010). Use of zona pellucida-bound sperm

Marchesi DE, Biederman H, Ferrara S, Hershlag A, Feng HL. (2010) The effect of semen

Markoulaki S, Kurokawa M, Yoon SY, Matson S, Ducibella T, Fissore R (2007) Comparison

Mauri AL, Petersen CG, Oliveire JB, Massaro FC, Baruffi LR, Franco JG Jr. (2010)

Menezo Y & Nicollet B. (2004) Replacement of PVP by Hyaluronate (SpermSlow™) in ICSI -

Menezo Y, Junca AM, Dumont-Hassan M, De Mouzon J, Cohen-Bacrie P, Ben Khalifa M.

Toluidine Blue Assay. *Eur J Obstet Gynecol Reprod Biol* 151, 176-180.

oocytes. *Eur J Obstet Gynecol Reprod Biol* 150, 42-46.

polyvinylpyrrolidone (PVP). *Fertil Steril* 94 (Supp 1): 232.

frequency of chromosomal aneuploidies. *Fertil Steril* 84, 1665-1673.

acrosomal status. *Fertil Steril* 79 (Suppl 3), 1616-1624.

aspects. *Reprod Biomed Online* 14, 650-663.

*Fertil Steril* 94, 2626-2630.

*Fertil Steril* 46, 1118-1123.

23,105-111.

815-818.

265-272.

binding by human sperm indicates cellular maturity, viability, and unreacted

testing and ICSI sperm selection by hyaluronic acid binding: clinical and genetic

Effects of semen storage and separation techniques on sperm DNA fragmentation.

Intracytoplasmic sperm injection: a novel selection method for sperm with normal

(1986) Sperm morphologic features as a prognostic factor in in vitro fertilization.

value of abnormal sperm morphology in in vitro fertilization. *Fertil Steril* 49, 112-117.

Follow-up of cognitive and motor development of 10-year-old singleton children born after ICSI compared with spontaneously conceived children. *Hum Reprod* 

for intracytoplasmic sperm injection produces higher embryo quality and implantation than conventional intracytoplasmic sperm injection. *Fertil Steril* 95,

processing on sperm DNA integrity: comparison of two techniques using the novel

of Ca2+ and CaMKII responses in IVF and ICSI in the mouse. *Mol Hum Reprod* 13,

Comparison of day 2 embryo quality after conventional ICSI versus intracytoplasmic morphologically selected sperm injection (IMSI) using sibling

Impact on outcome. Abstract of 18th World Congress on Fertility and Sterility IFFS.

(2010) ''Physiologic'' (hyaluronic acid-carried) icsi results in the same embryo quality and pregnancy rates as with the use of potentially toxic


**8**

*Tucson, Arizona* 

*Xi'an, Shaanxi* 

*2People's Republic of China* 

*1USA* 

**Advances in Fertility Options**

Bin Wu1, Timothy J. Gelety1 and Juanzi Shi2

*1ArizonaCenter for Reproductive Endocrinology and Infertility* 

*2Shaanxi Province Hospital for Women and Children Health Care* 

Infertility is defined as the failure of a couple to become pregnant after one year of regular, unprotected, sexual intercourse. Misconceptions about infertility are very common. Infertility affects roughly 15% of all couples during their reproductive lives. Infertility is not "just a female problem" as there is a male infertility component in approximately 50% of couples. A couple experiencing infertility should not underestimate the significance of the problems that can exist in the male. Male infertility may be the sole contributing reason for the couple's failure to conceive and should be best identified by a male infertility specialist. According to the 2009 national summary report of the American Society for Assisted Reproductive Technology, nearly 35% of infertility is attributed to the male factor. In the late last century, treatment for severe male factor infertility was limited to inseminations or *in vitro* fertilization using donor sperm. However, most infertile couples, and particularly men, are reluctant to use donor sperm because of bias maintained across cultural and ethnic boundaries. Today, exciting advances in male infertility have introduced innovative therapeutic options, in particular, intracytoplasmic sperm injection (ICSI), which offers men, including those with no sperm (azoospermia) in their ejaculate due to genetic conditions, a greatly improved chance to conceive their own biological offspring (Palermo, et al.. 1992). Currently, despite severe male factor infertility, pregnancy may still be achieved. This is mainly attributed to the success of the ICSI technique and advanced surgical testicular sperm retrieval techniques. In recent years, the ICSI technique has evolved into intracytoplasmic morphologically selected sperm injection (IMSI) and a method for selection of hyaluronan bound sperm for use in ICSI (PICSI) (Parmegiani et al., 2010a,b; Said & Land 2011; Berger et al., 2011), resulting in significantly increased fertilization and pregnancy rates over the world IVF clinics. Because the advanced ICSI technique in human IVF is covered in depth by other chapters of this book, the main emphasis here will be on the application of testicular biopsy sperm, round or elongated spermatids from azoospermic men to human IVF as well as explore new technologies to produce artificial sperm from

**1. Introduction** 

stem cells and somatic cells.

**of Azoospermic Men** 

