**Management of Pregnancy After Conization and Radical Trachelectomy**

Keun-Young Lee and Ji-Eun Song

 *Division of Maternal-Fetal Medicine, Department of Obstetrics and Gynecology, Hallym University, Korea*

## **1. Introduction**

Cervical intraepithelial neoplasia (CIN) is commonly diagnosed in women of child-bearing age. (Castle et al., 2009) The excisional treatments of CIN include cold-knife conization, laser conization, loop electrosurgical excision procedure (LEEP), and large loop excision of the transformation zone (LLETZ). CIN is also treated by ablative procedures such as laser vaporization and cryotherapy. Recently, human papilloma virus (HPV) has led to the understanding of pathogenesis and management of cervical diseases. (Moscicki et al., 2004) The screening and subsequent treatment of CIN significantly reduces the risk of invasive cervical cancer, but the impact of excisional cervical treatment on subsequent pregnancy outcome must be evaluated.

Many women with a diagnosis of cervical cancer are child-bearing population, younger than 40 years. Fertility-sparing radical vaginal or abdominal trachelectomy is a safe and feasible procedure for the treatment of patients with early stage cervical cancer. (Plante et al., 2005; Roman, 2005; Hertel et al., 2006) The disease-free survival rates and overall survival rates of radical trachelectomy is comparable to those of radical hysterectomy (Rob et al., 2011) The proper waiting period after radical tracelectomy and before conception varies in the literature. Roy and Plante (Roy and Plante, 1998) recommend a 6 month follow-up, while Shlaerth *et al*. (Shlaerth et al., 2003) describe a period of 1 year. The issue of having a pregnancy and fetal viability must be addressed following radical trachelectomy.

This chapter will outline concerns and management options of pregnancy after cervical surgeries with conization and radical trachelectomy.

## **2. Pregnancy after conization and radical trachelectomy**

The important issue following cervical surgery in child-bearing age is the fertility and pregnancy outcome. Excisional cervical surgery may contribute to decreased mechanical support of cervix, and increased susceptibility to ascending infection resulting from the loss of cervical mucus. Cervical surgery may alter reproductive function by cervical shortening, impairment of immunologic defence, and alteration of cervicovaginal bacterial flora, and thus expose those women to high risk of preterm birth. (Svare et al., 1992)

Management of Pregnancy After Conization and Radical Trachelectomy 327

Between 14 and 22 weeks of gestation, the median cervical length is 35-40 mm. From 22 to 32 weeks, the 50th percentile for length is 35 mm, and the 10th percentile is 25 mm. (Iam, 2004) A cervical length cutoff level of < 25 mm was associated with a sensitivity of 69%, a specificity of 80%, and a positive predictive value of 55%. (Owen et al., 2001) The <25 mm cutoff level before 20 weeks of gestation predicted preterm birth at <35 weeks gestation [likelyhood ratio (LR) 4.31, 95% CI 30.8-6.01, n =2258] (Crane and Hutchens, 2008) Independent of baseline cervical length, dynamic cervical change (ie, millimeters per week) was

Funneling is defined as the opening of the internal os of cervix. The significance of funneling on pregnancy after conization has not been reported. Compared with a short cervix (<25mm) alone, The combination of short cervix (<25mm) and funneling increased the sensitivity of predicting preterm delivery from 61% to 74 %. (Berghella et al., 1999) However, the normal cervical length more than 25 mm plus funneling does not increase the

Fetal fibronectin (FFN) is a widely accepted predictor of preterm birth. The presence of FFN in cervicovaginal secretions is significantly associated with preterm birth. (Peaceman et al., 1997) Women with a positive FFN and a short cervix (<22mm) had a significantly shorter

The uterine electromyography, which detects the electrical activity of myometrium, may be

The presence of bacterial vaginosis increases the risk of preterm delivery, (Leitich and Kiss, 2007) but no data suggests the correlation between vaginal infection and pregnancy outcome after conization. Bacterial vaginosis is further attributable to preterm birth in women with short cervix (<25mm). (Rust et al., 2005) There is no study that reveals whether other infection such as Chlamydia, or gonorrhea contributes to preterm delivery and

The lack of mechanical support of cervical mucus after radical trachelectomy may cause cervical incompetence and ascending infection. Shepherd et al (Shepherd et al., 2001) suggested the use of prophylactic antibiotics between 14 and 16 weeks of gestation following radical trachelectomy. Saling operation (complete cervicovaginal occlusion) which should be performed between 12 and 14 weeks of gestation may be the preventive strategy for reducing the risk of ascending infection and chorioamnionitis (Shlaerth et al., 2003**;**

Progesterone appears to prevent cervical ripening, (Word et al., 2007; Yellon et al., 2009) and anti-inflammatory properties. (Elovitz et al., 2008; Elovitz et al., 2005) The precise mechanisms of blockade of progesterone action against cervical changes are poorly

significantly associated with higher risk for preterm birth. (Owen et al., 2001)

prediction of preterm birth. (Berghella and Roman, 2005)

used to diagnose preterm labor. (Lucovnik et al., 2011)

**2.1.2 Nonsonographic methods**

latency to delivery. (Rizzo et al., 1996)

**2.1.3 Infection**

cervical length.

Gadducci et al., 2001)

**2.1.4 Progesterone**

understood.

Early studies on the impact of conization on pregnancy are hindered by small study population, inadequately powered study design, and neglect of potential confounders such as socioeconomic status and previous preterm delivery. (Sadler et al., 2007)Recent welldesigned studies with large sample sizes conclude that conization increases the risk of preterm birth and perinatal morbidities (Jakobsson et al., 2007; Sjoborg et al., 2007; Nohr et al., 2007) Jakobsson *et al*. (Jakobsson et al., 2007) found a significant increase of very preterm birth (28-31 weeks) and extremely preterm birth (< 28 weeks) following conization. [relative risk (RR) 1.99, 95% confidence interval (CI) 1.81-2.20; RR 2.86, 95% CI 1.30-2.32, respectively]. The study also showed a significant association of low birth weight (LBW) and conization.[RR 2.06, 95% CI 1.83-2.31] Other two meta-analyses also supported that excisional cervical treatments in CIN subsequently increase risk of preterm birth, and preterm premature rupture of membrane (PPROM) (Crane, 2003) Some studies reported the association between cone depth and higher risk of preterm birth, but the limited and conflicting data failed to demonstrate an accurate results. (Salder et al., 2004; Samson et al., 2005)

Because cervix is amputated below the cervical isthmus in radical trachelectomy, obstetric morbidity is higher than those of conization. The risk of preterm birth after radical trachelectomy increases two-fold than the general population. (Jolley et al., 2007; Martin et al., 2008) Boss *et al*. (Boss et al., 2005) reviewed obstetric outcomes of radical trachelectomy in the literature. A total of 335 cases of radical trachelectomy were analyzed. 153/355 (43%) patients attempted to become pregnant. About 30% of these women did not conceive. The causes of their infertility include anovulation, cervical factor such as cervical stenosis, and idiopathic factor. Other 70% of these women subsequently became pregnant. Of these, first and second trimester losses occurred in 47/161 (29%) pregnancies. 21% ended in the third trimester before 36 weeks of gestation. The causes of their preterm birth were attributed to cervical incompetence, ascending infection, and PPROM. (Boss et al., 2005)

#### **2.1 Management options**

It is clear that cervical surgeries attribute to increased risk of preterm birth (Kyrgiou et al., 2006; Plante et al., 2005; Samson et al., 2005; Sadler et al., 2004), but no specific rationale of pregnancy management after cervical surgeries exist. The management strategies for pregnancy after cervical surgeries should refer to those for women with short cervix who are at high risk for preterm birth.

## **2.1.1 Transvaginal ultrasound**

It is well known that women with a short cervix in the mid-trimester have a higher risk for preterm birth. (Iam et al., 1996; To et al., 2001) Cervical length, as determined by vaginal sonography, has been shown to be sensitive, and reproducible than digital examination or transabdominal ultrasonogrphy in predicting the risk of preterm birth. (Berghella and Bega, 2008) Cervical length is measured from the internal os to the external os. When cervical funneling is visualized, cervical length is measurable form the functional internal os to the external os. Some researchers reported the effect of conization on subsequent cervical length. Gentry et al. (Gentry et al., 2000) found no difference in cervical length between before and after LEEP. Berghella et al.(Berghella et al., 2004) demonstrated that only 28% of patients showed cervical shortening following conization.

Between 14 and 22 weeks of gestation, the median cervical length is 35-40 mm. From 22 to 32 weeks, the 50th percentile for length is 35 mm, and the 10th percentile is 25 mm. (Iam, 2004) A cervical length cutoff level of < 25 mm was associated with a sensitivity of 69%, a specificity of 80%, and a positive predictive value of 55%. (Owen et al., 2001) The <25 mm cutoff level before 20 weeks of gestation predicted preterm birth at <35 weeks gestation [likelyhood ratio (LR) 4.31, 95% CI 30.8-6.01, n =2258] (Crane and Hutchens, 2008) Independent of baseline cervical length, dynamic cervical change (ie, millimeters per week) was significantly associated with higher risk for preterm birth. (Owen et al., 2001)

Funneling is defined as the opening of the internal os of cervix. The significance of funneling on pregnancy after conization has not been reported. Compared with a short cervix (<25mm) alone, The combination of short cervix (<25mm) and funneling increased the sensitivity of predicting preterm delivery from 61% to 74 %. (Berghella et al., 1999) However, the normal cervical length more than 25 mm plus funneling does not increase the prediction of preterm birth. (Berghella and Roman, 2005)

## **2.1.2 Nonsonographic methods**

Fetal fibronectin (FFN) is a widely accepted predictor of preterm birth. The presence of FFN in cervicovaginal secretions is significantly associated with preterm birth. (Peaceman et al., 1997) Women with a positive FFN and a short cervix (<22mm) had a significantly shorter latency to delivery. (Rizzo et al., 1996)

The uterine electromyography, which detects the electrical activity of myometrium, may be used to diagnose preterm labor. (Lucovnik et al., 2011)

## **2.1.3 Infection**

326 Hysterectomy

Early studies on the impact of conization on pregnancy are hindered by small study population, inadequately powered study design, and neglect of potential confounders such as socioeconomic status and previous preterm delivery. (Sadler et al., 2007)Recent welldesigned studies with large sample sizes conclude that conization increases the risk of preterm birth and perinatal morbidities (Jakobsson et al., 2007; Sjoborg et al., 2007; Nohr et al., 2007) Jakobsson *et al*. (Jakobsson et al., 2007) found a significant increase of very preterm birth (28-31 weeks) and extremely preterm birth (< 28 weeks) following conization. [relative risk (RR) 1.99, 95% confidence interval (CI) 1.81-2.20; RR 2.86, 95% CI 1.30-2.32, respectively]. The study also showed a significant association of low birth weight (LBW) and conization.[RR 2.06, 95% CI 1.83-2.31] Other two meta-analyses also supported that excisional cervical treatments in CIN subsequently increase risk of preterm birth, and preterm premature rupture of membrane (PPROM) (Crane, 2003) Some studies reported the association between cone depth and higher risk of preterm birth, but the limited and conflicting data failed to demonstrate an accurate results. (Salder et al., 2004; Samson et al.,

Because cervix is amputated below the cervical isthmus in radical trachelectomy, obstetric morbidity is higher than those of conization. The risk of preterm birth after radical trachelectomy increases two-fold than the general population. (Jolley et al., 2007; Martin et al., 2008) Boss *et al*. (Boss et al., 2005) reviewed obstetric outcomes of radical trachelectomy in the literature. A total of 335 cases of radical trachelectomy were analyzed. 153/355 (43%) patients attempted to become pregnant. About 30% of these women did not conceive. The causes of their infertility include anovulation, cervical factor such as cervical stenosis, and idiopathic factor. Other 70% of these women subsequently became pregnant. Of these, first and second trimester losses occurred in 47/161 (29%) pregnancies. 21% ended in the third trimester before 36 weeks of gestation. The causes of their preterm birth were attributed to

It is clear that cervical surgeries attribute to increased risk of preterm birth (Kyrgiou et al., 2006; Plante et al., 2005; Samson et al., 2005; Sadler et al., 2004), but no specific rationale of pregnancy management after cervical surgeries exist. The management strategies for pregnancy after cervical surgeries should refer to those for women with short cervix who

It is well known that women with a short cervix in the mid-trimester have a higher risk for preterm birth. (Iam et al., 1996; To et al., 2001) Cervical length, as determined by vaginal sonography, has been shown to be sensitive, and reproducible than digital examination or transabdominal ultrasonogrphy in predicting the risk of preterm birth. (Berghella and Bega, 2008) Cervical length is measured from the internal os to the external os. When cervical funneling is visualized, cervical length is measurable form the functional internal os to the external os. Some researchers reported the effect of conization on subsequent cervical length. Gentry et al. (Gentry et al., 2000) found no difference in cervical length between before and after LEEP. Berghella et al.(Berghella et al., 2004) demonstrated that only 28% of

cervical incompetence, ascending infection, and PPROM. (Boss et al., 2005)

2005)

**2.1 Management options**

are at high risk for preterm birth.

**2.1.1 Transvaginal ultrasound**

patients showed cervical shortening following conization.

The presence of bacterial vaginosis increases the risk of preterm delivery, (Leitich and Kiss, 2007) but no data suggests the correlation between vaginal infection and pregnancy outcome after conization. Bacterial vaginosis is further attributable to preterm birth in women with short cervix (<25mm). (Rust et al., 2005) There is no study that reveals whether other infection such as Chlamydia, or gonorrhea contributes to preterm delivery and cervical length.

The lack of mechanical support of cervical mucus after radical trachelectomy may cause cervical incompetence and ascending infection. Shepherd et al (Shepherd et al., 2001) suggested the use of prophylactic antibiotics between 14 and 16 weeks of gestation following radical trachelectomy. Saling operation (complete cervicovaginal occlusion) which should be performed between 12 and 14 weeks of gestation may be the preventive strategy for reducing the risk of ascending infection and chorioamnionitis (Shlaerth et al., 2003**;**  Gadducci et al., 2001)

#### **2.1.4 Progesterone**

Progesterone appears to prevent cervical ripening, (Word et al., 2007; Yellon et al., 2009) and anti-inflammatory properties. (Elovitz et al., 2008; Elovitz et al., 2005) The precise mechanisms of blockade of progesterone action against cervical changes are poorly understood.

Management of Pregnancy After Conization and Radical Trachelectomy 329

conization. (Berghella et al., 2005) A short cervix in women with prior cervical surgery are predictive of preterm delivery, but the attributable risk is lower than in women with index preterm birth. More recently, the Vaginal Ultrasound Trail Consortium (Owen J,2008) demonstrated that the effect of cerclage for preventing preterm birth and prolonging gestation was identified only in women with a cervical length of less than 15 mm (RR 0.23, 95% CI 0.08 – 0.66). Zeisler et al (Zeisler et al., 1997) specifically evaluated the cerclage in women with prior conization, and concluded that prophylactic cerclage is not beneficial for preventing preterm birth. The combination of present cervical length, and prior histories of preterm birth and cervical surgeries is a pronounced risk factor of preterm delivery. Once the cervix is shortened, it is difficult to place satisfactory McDonald cerclage. Surgeons

Physical-examination based cerclage is placed in women with significant cervical dilatation (>1cm) and bulging membrane. It is referred to as a 'rescue' or 'emergency' cerclage, as it requires challenging skill to avoid a rupture of membrane during cerclage. Daskalakis et al. (Daskalakis et al., 2006) concluded that physical-examination cerclage reduced preterm delivery before 32 weeks of gestation and improved neonatal survival rate compared to bed rest group. Other researchers (Cockwell and Smith, 2005) also demonstrated that physicalexamination cerclage prolonged pregnancy and improved neonatal outcome in women with dilated cervix and bulging membrane. Lee et al (Lee et al., 2004) reported that elevated interleukin-6 in amniotic fluid was adversely associated with interval between physicalexamination cerclage and delivery. Weiner et al. (Weiner et al., 2005) found intrauterine inflammatory responses and decidual hemorrhage were correlated with adverse pregnancy outcome after physical-examination cerclage. A physical-examination cerclage could be considered in women with dilated cervix and bulging membrane after conization. In these women, the evaluation of subclinical inflammation may predict the prognosis of pregnancy. Transabodminal cervico-isthmic cerclage (TCIC) is beneficial in women with cervix that is extremely short, congenital anomaly or markedly scarred due to previous vaginal cerclage. TCIC seems to be effective in women who are inadequate for vaginal cerclage. (Topping et al., 1995; Turnquest et al., 1999) The unrelenting complication during pregnancy following radical trachelectomy is late abortion and preterm delivery as cervix has markedly modified anatomy after radical trachelectomy. The prophylactic concomitant cerclage is usually placed at the time of radical trachelectomy. (Plante et al., 2005; Roman, 2005) When the cerclage placed too deeply within the cervical stroma, it can be expelled spontaneously. The success of pregnancy may depends on a stable maintenance of cerclage. (Plante et al., 2005) Transabdominal cervico-isthmic cerclage is an appreciable option for the pregnant women after radical trachelectomy because of lack of sufficient cervical tissue for vaginal cerclage.

Preterm delivery in twin pregnancies is 5-10 time more frequent than that in singleton pregnancy. (Fuchs et al., 2004) There is no clear best cervical length for the prediction of preterm birth in multiple pregnancies. Nevertheless, the rate of delivery was inversely associated with cervical length in twin pregnancies, being 66% for 10 mm, 24% for 20 mm,

should avoid bladder or bowel injury during ultrasound-indicated cerclage.

(Lee et al., 2007)

**2.1.6 Multiple pregnancies**

12% for 25 mm, and < 1% for 40 mm. (To et al, 2006).

Fonesca et al (Fonesca et al, 2007) specifically evaluated the impact of progesterone in women with a short cervix (<15mm) identified on transvaginal ultrasonography. Women were allocated to receive either 200 mg nightly intravaginal progesterone or placebo from 24 to 33 weeks of gestation. A total of 250 women were enrolled. Women administered progesterone less likely to have a spontaneous delivery before 34 weeks of gestation than placebo group. (19.2 % vs 34.4 %; relative risk [RR] = 0.56; 95% confidence interval [CI] = 0.36-0.86). There was a significant reduction in the risk of neonatal sepsis in women administered progesterone (274 infants, RR = 0.28, 95% CI 0.08 – 0.97). (Fonesca et al, 2007) A systematic meta-analysis by Dodd et al (Dodd et al., 2008) also demonstrated that vaginal progesterone significantly reduced preterm birth before 34 weeks of gestation in women with short cervix. Facchinetti et al (Facchinetti et al, 2007) reported a randomized clinical trial involving symptomatic women with preterm labor and intact membranes at 25- 34 weeks of gestation. For women administered intramuscular 341mg of 17-alphahydroxyprogesterone caproate (17P) twice a week until 36 weeks, when compared with observation group, there was a significant reduction in the risk of spontaneous delivery. (odds ratio [OR] = 0.15, 95% CI = 0.04-0.58) The subgroup analysis involving women with short cervix less than 25 mm at enrolment showed more significant difference between two groups. Women administered 17P had less cervical shortening at days 21 (1.38 ± 1.31 mm) compared to women who had not received 17P (day 21, 4.88 ± 3.14 mm, p = 0.0001) (Facchinetti et al, 2007) Future studies are needed to enlighten the association between prophylactic progesterone and pregnancy outcome after conization and radical trachelectomy.

#### **2.1.5 Cerclage**

There is considerable debate as to whether cerclage is efficient in pregnant women after conization. The exact mechanism of preterm birth after conization is unclear, and so the mechanisum of cervical shortening following conization is unclear.

Royal College of Obstetrics and Gynecology (RCOG, 1993) performed a trial of historyindicated cerclage (prophylactic cerclage). A total of 1292 women were enrolled and 138 women with a history of conization or cervical amputation were included among them. The subgroup analysis of those 138 women revealed no significant reduction in preterm birth or improvement in neonatal outcome. However, there was a significant reduction in delivery less than 32 weeks of gestataion in cerclage group with 3 or more second-trimester deliveries, and no histories of cervical surgeries. (RCOG, 1993) Other researchers also reported that a prophylactic cerclage was not beneficial for preventing preterm birth in women with prior conization. (Nam et al., 2010; Shin et al., 2010)

The transvaginal sonography of cervix enables obstetricians to detect cervical shortening or funneling in asymptomatic women. Ultrasound-indicated cerclage based on a short cervix has been proposed as a solution for preventing cervical incompetence and preterm birth. A meta-anaylsis (Berghella et al., 2005) including four randomized trials of ultrasoundindicated cerclage found that cerclage is beneficial in women with short cervix less than 25 mm before 24 weeks of gestation and index preterm birth. In these women, a relative risk reduction of preterm birth at < 35 weeks of gestation was enhanced. (RR, 0.61; 95% ci, 0.4- 0.9) In the subgroup analysis, there was no beneficial effect of cerclage on women with prior

Fonesca et al (Fonesca et al, 2007) specifically evaluated the impact of progesterone in women with a short cervix (<15mm) identified on transvaginal ultrasonography. Women were allocated to receive either 200 mg nightly intravaginal progesterone or placebo from 24 to 33 weeks of gestation. A total of 250 women were enrolled. Women administered progesterone less likely to have a spontaneous delivery before 34 weeks of gestation than placebo group. (19.2 % vs 34.4 %; relative risk [RR] = 0.56; 95% confidence interval [CI] = 0.36-0.86). There was a significant reduction in the risk of neonatal sepsis in women administered progesterone (274 infants, RR = 0.28, 95% CI 0.08 – 0.97). (Fonesca et al, 2007) A systematic meta-analysis by Dodd et al (Dodd et al., 2008) also demonstrated that vaginal progesterone significantly reduced preterm birth before 34 weeks of gestation in women with short cervix. Facchinetti et al (Facchinetti et al, 2007) reported a randomized clinical trial involving symptomatic women with preterm labor and intact membranes at 25- 34 weeks of gestation. For women administered intramuscular 341mg of 17-alphahydroxyprogesterone caproate (17P) twice a week until 36 weeks, when compared with observation group, there was a significant reduction in the risk of spontaneous delivery. (odds ratio [OR] = 0.15, 95% CI = 0.04-0.58) The subgroup analysis involving women with short cervix less than 25 mm at enrolment showed more significant difference between two groups. Women administered 17P had less cervical shortening at days 21 (1.38 ± 1.31 mm) compared to women who had not received 17P (day 21, 4.88 ± 3.14 mm, p = 0.0001) (Facchinetti et al, 2007) Future studies are needed to enlighten the association between prophylactic progesterone and pregnancy outcome after conization and radical

There is considerable debate as to whether cerclage is efficient in pregnant women after conization. The exact mechanism of preterm birth after conization is unclear, and so the

Royal College of Obstetrics and Gynecology (RCOG, 1993) performed a trial of historyindicated cerclage (prophylactic cerclage). A total of 1292 women were enrolled and 138 women with a history of conization or cervical amputation were included among them. The subgroup analysis of those 138 women revealed no significant reduction in preterm birth or improvement in neonatal outcome. However, there was a significant reduction in delivery less than 32 weeks of gestataion in cerclage group with 3 or more second-trimester deliveries, and no histories of cervical surgeries. (RCOG, 1993) Other researchers also reported that a prophylactic cerclage was not beneficial for preventing preterm birth in

The transvaginal sonography of cervix enables obstetricians to detect cervical shortening or funneling in asymptomatic women. Ultrasound-indicated cerclage based on a short cervix has been proposed as a solution for preventing cervical incompetence and preterm birth. A meta-anaylsis (Berghella et al., 2005) including four randomized trials of ultrasoundindicated cerclage found that cerclage is beneficial in women with short cervix less than 25 mm before 24 weeks of gestation and index preterm birth. In these women, a relative risk reduction of preterm birth at < 35 weeks of gestation was enhanced. (RR, 0.61; 95% ci, 0.4- 0.9) In the subgroup analysis, there was no beneficial effect of cerclage on women with prior

mechanisum of cervical shortening following conization is unclear.

women with prior conization. (Nam et al., 2010; Shin et al., 2010)

trachelectomy.

**2.1.5 Cerclage**

conization. (Berghella et al., 2005) A short cervix in women with prior cervical surgery are predictive of preterm delivery, but the attributable risk is lower than in women with index preterm birth. More recently, the Vaginal Ultrasound Trail Consortium (Owen J,2008) demonstrated that the effect of cerclage for preventing preterm birth and prolonging gestation was identified only in women with a cervical length of less than 15 mm (RR 0.23, 95% CI 0.08 – 0.66). Zeisler et al (Zeisler et al., 1997) specifically evaluated the cerclage in women with prior conization, and concluded that prophylactic cerclage is not beneficial for preventing preterm birth. The combination of present cervical length, and prior histories of preterm birth and cervical surgeries is a pronounced risk factor of preterm delivery. Once the cervix is shortened, it is difficult to place satisfactory McDonald cerclage. Surgeons should avoid bladder or bowel injury during ultrasound-indicated cerclage.

Physical-examination based cerclage is placed in women with significant cervical dilatation (>1cm) and bulging membrane. It is referred to as a 'rescue' or 'emergency' cerclage, as it requires challenging skill to avoid a rupture of membrane during cerclage. Daskalakis et al. (Daskalakis et al., 2006) concluded that physical-examination cerclage reduced preterm delivery before 32 weeks of gestation and improved neonatal survival rate compared to bed rest group. Other researchers (Cockwell and Smith, 2005) also demonstrated that physicalexamination cerclage prolonged pregnancy and improved neonatal outcome in women with dilated cervix and bulging membrane. Lee et al (Lee et al., 2004) reported that elevated interleukin-6 in amniotic fluid was adversely associated with interval between physicalexamination cerclage and delivery. Weiner et al. (Weiner et al., 2005) found intrauterine inflammatory responses and decidual hemorrhage were correlated with adverse pregnancy outcome after physical-examination cerclage. A physical-examination cerclage could be considered in women with dilated cervix and bulging membrane after conization. In these women, the evaluation of subclinical inflammation may predict the prognosis of pregnancy.

Transabodminal cervico-isthmic cerclage (TCIC) is beneficial in women with cervix that is extremely short, congenital anomaly or markedly scarred due to previous vaginal cerclage. TCIC seems to be effective in women who are inadequate for vaginal cerclage. (Topping et al., 1995; Turnquest et al., 1999) The unrelenting complication during pregnancy following radical trachelectomy is late abortion and preterm delivery as cervix has markedly modified anatomy after radical trachelectomy. The prophylactic concomitant cerclage is usually placed at the time of radical trachelectomy. (Plante et al., 2005; Roman, 2005) When the cerclage placed too deeply within the cervical stroma, it can be expelled spontaneously. The success of pregnancy may depends on a stable maintenance of cerclage. (Plante et al., 2005) Transabdominal cervico-isthmic cerclage is an appreciable option for the pregnant women after radical trachelectomy because of lack of sufficient cervical tissue for vaginal cerclage. (Lee et al., 2007)

#### **2.1.6 Multiple pregnancies**

Preterm delivery in twin pregnancies is 5-10 time more frequent than that in singleton pregnancy. (Fuchs et al., 2004) There is no clear best cervical length for the prediction of preterm birth in multiple pregnancies. Nevertheless, the rate of delivery was inversely associated with cervical length in twin pregnancies, being 66% for 10 mm, 24% for 20 mm, 12% for 25 mm, and < 1% for 40 mm. (To et al, 2006).

Management of Pregnancy After Conization and Radical Trachelectomy 331

Anonymous: Final report of the Medical Research Council/Royal College of Obstetrics and

Berghella V, Bega G: Ultrasound evaluation of ther cervix, in Callen PW (ed):

Berghella V, Dalsy SF, Tolosa JE, et al. Prediction of preterm delivery with tranvaginal

Berghella V, Odibo AO, To MS, et al. Cerclage for short cervix on ultrasound:meta-analysis of trials using individual patient-level data. Obstet Gynecol 2005;106:181-189 Berghella V, Pereira L, GariepyA, Simonazzi G. Prior cone biopsy: prediction of preterm birth by cervical ultrasound. Am J Obstet Gynecol 2004;191:1393-1397 Berghella V, Roman A. Does funneling increase the incidence of preterm birth in women with normal cervical length? Am J Obstet Gynecol 193;S147,2005(abstr) Boss EA, van Golde RJ, Beerendonk CC, Massuger LF. Pregnancy after radical trachelectomy: a real option. Gynecol Oncol 2005;99(Suppl 1):S152-6 Castle PE, Fetterman , Akhtar I, Husain M et al. Age-appropriate use of human papillomavirus vaccines in the U.S. Gynecol Oncol 2009;114(2):365-9 Cockwell HA, Smith GN.2005. Cervical incompetence and the role of emergency cerclage.

Crane JMG: Pregnancy outcome after loop electrosurgical excision procedure: A systematic

Crane JM, Hutchens D. Transvaginal sonogrpahic measurement of cervical length to predict

Daskalakis G, Papantoniou N, Mesogitis S, Antasaklis A. 2006. Management of cervical

Dodd JM, Flenady VJ, Cincotta R: Progesterone for the prevention of preterm birth: a

Elovitz MA, Gonzalez J : Medroxyprogesterone acetate modulates the immune response in

Elovitz MA, Mrinalini C Sammel MD: Can medroxyprogesterone acetate alter Toll-like

Eskandar M, Shafiq H, Almushait MA, et al. Cervical cerclage for prevention of preterm birth in women with twin pregnancy. Int Gynecol Obstet 2007;99:110-112 Facchinetti F, Paganelli S, Comitini G, et al: Cervical length changes during preterm cervical

Fonesca EB, Celik E, Parra M, Singh M, Nicolaides KH, for the Fetal Medicine Foundation

among women with a short cervix. N Engl J Med 2007;357:462-9

preterm birth in astymptomatic women at increased risk: a systematic review.

insufficiency and bulging fetal membranes. Obstetrics and Gynecology 107:221-226

the uterus, cervix and placenta in a mouse model of preterm birth. J Matern Fetal

receptor expression in a mouse model of intrauterine inflammation? Am J Obstet

ripening: effects of 17-alpha-hydroxyprogesterone caproate. Am J Obstet Gynecol

Second Trimster Screening Group. Progesterone and the risk of preterm birth

prevent prematurity? Am J Obstet Gynecol 181:809-815,1999

Journal of Obstetrics and Gynecology Canada 27:123-129

review. Obstet Gynecol 102 (2003) 1058

Neonatal Med 21:223-230,2008

Gynecol 193:1149-1155, 2005

196:453-54,2007

Ultrasound Obstet Gynecol 2008;31:579-587

systematic review. Obstet Gynecol 112:127-134, 2008

Gynecology multicenter randomized trial of cervical cerclage. Br J Obstet Gynecol

Ultrasonography in Obstetrics and Gynecology (ed 5). Philadelphia, PA, Saunders

ultrasonography of the cervix in patients with high-risk pregnancies: does cerclage

**5. References**

100:516-523,1993

Elsevier, 2008, pp 698-720

Current evidence does not support prophylactic administration of progesterone in multiple pregnancies. Additional studies are needed to confirm the effect of progesterone supplementation in twin pregnancies.

There is no data that prophylactic cerclage should be performed in multiple pregnancies. Recent prospective non-randomized trial of prophylactic cerclage in asymptomatic twin pregnancies reported that cerclage is not beneficial. (Eskandar et al., 2007) Twin pregnancies following radical trachelectomy are at more increased risk for preterm birth compared to singleton pregnancies. (Plante et al., 2005) Lee et al (Lee et al., 2007) reported TCIC after radical trachelectomy in twin pregnancy improved the pregnancy outcome. Cerclage in twin pregnancy is effective in certain circumstances.

## **3. Conclusion**

CIN is relatively common in women with child-bearing age. Several studies have reported that women undergoing conization are at increased risk for preterm delivery, a low birthweight neonate, and premature rupture of membrane. Recently, the conservative treatment of early cervical cancer to preserve fertility has improved. A cervix changes drastically after radical trachelectomy. In these women after conization or radical trachelectomy, obstetricians should consider the possible adverse effects on future pregnancies.

As noted earlier, there is no established rationale for the pregnancy management after conization and radical trachelectomy. There are several management options for preventing preterm birth in these women but none has been prove more effective than any other strategies: Transvaginal sonography is a sensitive and reproducible method to detect short cervix. The administration of progesterone is thought to have beneficial effect on prolonging pregnancy. History-indicated cerclage in women with previous conization seems to be inefficient in preventing preterm birth. Ultrasound-indicated cerclage could be beneficial in women with short cervix and a prior preterm birth history. Physical-examination cerclage could prevent preterm birth in women with dilated cervix and bulging membrane after cervical surgeries. TCIC is an acceptable option for pregnant women after radical trachelectomy and women with severely scarred cervix. The combination of above mentioned options and other adjunctive strategies such as detection of infection or inflammation, and antibiotics, etc. could reduce the rate of preterm birth in pregnancy after conization or radical trachelectomy.

Further studies are needed to assess an accurate measurement of cone tissue, and explore the relation between the amount of cone tissue and risk of deleterious pregnancy outcome.

Randomized clinical trials are needed to confirm the role of cerclage, and progesterone, and adjunctive therapies such as antibiotics, and in pregnant women with prior cervical surgeries.

#### **4. Acknowledgments**

We would like to thank all the members of the department of obstetrics and gynecology, Hallym University Medical Center.

## **5. References**

330 Hysterectomy

Current evidence does not support prophylactic administration of progesterone in multiple pregnancies. Additional studies are needed to confirm the effect of progesterone

There is no data that prophylactic cerclage should be performed in multiple pregnancies. Recent prospective non-randomized trial of prophylactic cerclage in asymptomatic twin pregnancies reported that cerclage is not beneficial. (Eskandar et al., 2007) Twin pregnancies following radical trachelectomy are at more increased risk for preterm birth compared to singleton pregnancies. (Plante et al., 2005) Lee et al (Lee et al., 2007) reported TCIC after radical trachelectomy in twin pregnancy improved the pregnancy outcome. Cerclage in twin

CIN is relatively common in women with child-bearing age. Several studies have reported that women undergoing conization are at increased risk for preterm delivery, a low birthweight neonate, and premature rupture of membrane. Recently, the conservative treatment of early cervical cancer to preserve fertility has improved. A cervix changes drastically after radical trachelectomy. In these women after conization or radical trachelectomy, obstetricians should consider the possible adverse effects on future

As noted earlier, there is no established rationale for the pregnancy management after conization and radical trachelectomy. There are several management options for preventing preterm birth in these women but none has been prove more effective than any other strategies: Transvaginal sonography is a sensitive and reproducible method to detect short cervix. The administration of progesterone is thought to have beneficial effect on prolonging pregnancy. History-indicated cerclage in women with previous conization seems to be inefficient in preventing preterm birth. Ultrasound-indicated cerclage could be beneficial in women with short cervix and a prior preterm birth history. Physical-examination cerclage could prevent preterm birth in women with dilated cervix and bulging membrane after cervical surgeries. TCIC is an acceptable option for pregnant women after radical trachelectomy and women with severely scarred cervix. The combination of above mentioned options and other adjunctive strategies such as detection of infection or inflammation, and antibiotics, etc. could reduce the rate of preterm birth in pregnancy after

Further studies are needed to assess an accurate measurement of cone tissue, and explore the relation between the amount of cone tissue and risk of deleterious pregnancy outcome. Randomized clinical trials are needed to confirm the role of cerclage, and progesterone, and adjunctive therapies such as antibiotics, and in pregnant women with prior cervical

We would like to thank all the members of the department of obstetrics and gynecology,

supplementation in twin pregnancies.

conization or radical trachelectomy.

**3. Conclusion**

pregnancies.

surgeries.

**4. Acknowledgments**

Hallym University Medical Center.

pregnancy is effective in certain circumstances.

Management of Pregnancy After Conization and Radical Trachelectomy 333

Nohr B, Tabor A, Fredericksen K, et al. Loop electrosurgical excision of the cervix and the subsequent risk of preterm delivery. Acta Obstet Gynecol Scand 2007;86:596-603 Owen J, Yost N, Berghella V, Thom E, Swain M, Dildy GA et al. Mid-trimester endogavinal

Owen J for the vagnial ultrasound trial consortium: Multicenter randomized trial of cerclage

Peaceman AM, Andrews WW, Thorp JM, et al: Fetal fibronectin as a predictor of PTB in patients with symptoms: a multicenter trial. Obstet Gynecol 177:13-18,1997 Plante M, Renaud MC, Hoskins IA, Roy M. Vaginal radical trachelectomy: a valuable

Rizzo G, Capponi A, Arduini D, et al: The value of fetal fibronectin in cervical and vaginal

Rob L, Skapa P, Robova H. Fertility-sparing surgery in patients with cervical cancer. Lancet

Roman LD. Pregnancy after radical vaginal tracelectomy: maybe not such a risky

Roy M, Plante M. Pregnancies after radical vaginal tracelectomy for early-stage cervical

Rust OA, Atlas RO, Kimmel S, et al. Does the presence of a funnel increase the risk of

Salder L, A Saftlas, W Wang, M Exeter, J Whittaker, L McCowan: Treatment for cervical

Samson SA, JR Bentley, TJ Fahey, DJ McKay, GH Gill: The effect of loop electrosurgical

Shepherd JH, Mould T, Oram DH. Radical trachelectomy in early stage carcinoma of the

Shin MY, Seo ES, Choi SJ, Oh SY, Kim BF, Bae DS, et al. The role of prophylactic cerclage in

Shlaerth JB, Spirtos NM, Shlaerth AC. Radical trachelectomy and pelvic lymphadenectomy

Sjoborg KD, Vistad I, Myrh SS, et al. Preganancy outcome after cervical cone excision: a

Svare JA, LF Anderson, J Langhoff-Roos, ET Jensen, B Bruun, I Lind, H Madsen: The relation

case-control study. Acta Obstet Gynecol Scand 2007;86:423-428

Sadler L, Saftlas A. Cervical surgery and preterm birth. J Perinat Med 2007;35:5-9

adverse perinatal outcome in a patient with a short cervix? Am J Obstet Gynecol

intraepithelial neoplasia and risk of preterm delivery. J Am Med Assoc 29(2004)

excision procedure on future pregnancy outcome. Obstet Gynecol 105(2005) 325-32

cervix: outcomes as judged by recurrence and fertility rates. Br J Obstet Gynaecol

preventing preterm delivery after electrosurgical conization. J Gynecol Oncol

with uterine preservation in the treatment of cervical cancer. Am J Obstet Gynecol

ship between prior cervical conisation, cercial microbial colonization and preterm

cervical length. Am J Obstet Gynecol 199;S3, 2008(6, suppl A)

2001;286:1340-8

Obstet Gynecol 175:1146-1151, 1996

undertaking after all. Gynecol Oncol 2005 Jul; 98: 1-2

cancer. Am J Obstet Gynecol 1998;179:1491-6

Oncol 2011;12:192-200

192:1060-1066, 2005

2100-6

2001;108:882-5

2010;21:230-6

2003;188(1):29-34

10

sonography in women at high risk for spontaneous preterm birth. JAMA

for preterm birth prevention in high-risk women with shortened mid-trimester

fertility-preserving option in the management of early-stage cervical cancer. A series of 50 pregnancies and review of the literature. Gynecol Oncol 2005 Jul; 98:3-

secretions and of ultrasonographic examination of the uterine cervix in predicting premature delivery for patients with preterm labor and intact membranes. Am J

Fuchs IB, Tsoi E, Herich W, et al; Sonographic measurement of cervical length in twin

Gadducci A, Cosio S, Cionini L, Genazzani AR. Neoadjuvant chemotherapy and concurrent

Gentry DJ, Baggish MS, Brady K, et al: The effects of loop excision of the transformation

Hertel H, Kohler C, Grund D, Hillemanns P,Possover M, Michels W, Schneider A. Radical

Iams JD, Goldenberg RL, Meis PJ, Mercer BM, Moawad A, Das A, Thom E, McNellis D,

Iam JD. 2004. Abnormal cervical competence. In: Creasy RK, Resnik R, Iams JD, editors.

Jakobsson M, Gissler M, Sainio S, et al. Preterm delivery after surgical treatment for cervical

Jolley JA, Battista L, Wing DA. Management of pregnancy after radical trachelectomy: case reports and systematic review of the literature. Am J Perinatol 2007;24:531-540 Kyrgiou M, G Koliopoulos, P Marin-Hirsch, M Arbyn, W Prendiville, E Paraskevaidis:

Lee KY, Jun HA, Roh JW, Song JE. Successful twin pregnancy after vaginal radical

Leitich H, Kiss H. Asymptomatic bacterial vaginosis and intermediate flora as risk factors

Lucovnik M, KuonRJ, Chambliss LR, et al. Use of uterine electromyography to diagnose term and preterm labor. Acta Obstet Gynecol Scand. 2011;90(2):150-157 Martin JA, Kung HC, Mathews TJ, et al. Annual summary of vital statistics: 2006.  Pregnancy outcome after cervical conization: risk

intra-epitelial lesions in young women. Lacet 354 (2004) 1678

intraepithelial neoplaisa. Obstet Gynecol 2007;109:309-313

Obsteterics and Gynecology 191:784-789

cervical cancer. Gynecol Oncol 2006;103:506-11

2001;21:3525-33

2000;182:516-20

Saunders. Pp 603-622.

2007;197(3):e5-6

2008;121:788-801

Oncol 2010;21:225-9

390

572

pregnancies in threatened preterm labor. Ultrasound Obstet Gynecol 23:42-45,2004

chemoradidation in the treatment of advanced cervical cancer. Anticancer Res

zone on cervical length: implications for pregnancy. Am J Obstet Gynecol

vaginal trachelectomy (RVT) combined with laparoscopic pelvic lymphadenectomy: prospective multincenter study of 100 patients with early

Copper RL, Johnson F, Roberts JM. The length of the cervix and the risk of spontaneous premature delivery. National Institute of Child Health and Human Development Maternal Fetal Medicine Unit Network. N Engl J Med 1996;334:567-

Maternal-fetal medicine: principals and practice. 5th ed. Philadelphia: W B

Obstetric outcomes after conservative treatment for intraepithelial or early invasive cervical lesions: systematic review and meta-analysis. Lancet 367(2006)489-98 Lee KY, Jun HA, Kim HB, Kang SW.2004. Interleukin-6, but not relaxin, predicts outcome of

rescue cerclage in women with cervical incompetence. American Journal of

trachelectomy using trasabdominal cervicoisthmic cerclage. Am J Obstet Gynecol

for adverse pregnancy outcome. Best Pract Res Clin Obstet Gynaecol 2007; 21:375-

Clayton, S Farhat, JM Broe ring TM Darragh: Regression of low-grade squamous

factors for preterm delivery and the efficacy of prophylactic cerclage. J Gynecol

**22**

*South Korea*

**Know-How of the Hormonal Therapy and**

**the Effect of the Male Hormone on Uterus**

Management of the patient with gender identity disorder involves many different aspects of medical care. Transsexualism is a multidimensional phenomenon that requires a multidisciplinary approach in both the diagnostic and treatment phases. Endocrinologists have to work together with mental health professionals and surgeons to provide full counselling, confirm the diagnosis and avoid mistakes or regrets. Hormonal therapy not

Trends in Europe are toward treatment of patients at early age if the diagnosis is appropriately established. This may begin with blocking agents that temporarily suppress pubertal transformation. Secondary sexual trait suppression allows much more successful

Hormonal therapy is used to suppress secondary sexual characteristic of the biological sex, to induce the secondary sex characteristics of the new sex. This therapy should continue

Endocrinologic masculinization is achieved by the use of testosterone to induce male physical characteristics. Testosterone works primarily by direct stimulation of receptors in target tissues. It elevates serum testosterone level to a male reference range rather than a decrease in serum estradiol[2]. Testosterone also has antigonadotropic action in high

Rapidity and degree of change from testosterone therapy depends on the agents used, dosage, and the patient's responsiveness to endocrine therapy. Typically, within the first 1-3 months patients experience oilier skin and acne, increased libido, increased muscle mass and upper body strength, and redistribution of the fat to a more masculine

only has a therapeutic role but also an important diagnostic tool.

**2. Masculinizing effect in female to male hormonal therapy**

**1. Introduction**

gender transformation.

lifelong[1].

doses.

pattern[3, 12].

**in the Female to Male Transsexuals**

Seok Kwun Kim1 and Myoungseok Han2 *1Department of Plastic and Reconstructive Surgery,*

> *2Department of Obstetrics and Gynecology, Dong-A University School of Medicine,*

premature rupture of the membranes. Eur J Obstet Gynecol Repord Biol 47 (1992) 41


**1. Introduction**

Retinal imaging is limited due to optical aberrations caused by imperfections in the optical media of the eye. Consequently, diffraction limited retinal imaging can be achieved if optical aberrations in the eye are measured and corrected. Information about retinal pathology and structure on a cellular level is thus not available in a clinical setting but only from histologi‐ cal studies of excised retinal tissue. In addition to limitations such as tissue shrinkage and distortion, the main limitation of histological preparations is that longitudinal studies of dis‐ ease progression and/or results of medical treatment are not possible.

Adaptive optics (AO) is the science, technology and art of capturing diffraction-limited im‐ ages in adverse circumstances that would normally lead to strongly degraded image quality and loss of resolution. In non-military applications, it was first proposed and implemented in astronomy [1]. AO technology has since been applied in many disciplines, including vi‐ sion science, where retinal features down to a few microns can be resolved by correcting the aberrations of ocular optics. As the focus of this chapter is on AO retinal imaging, we will focus our description to this particular field.

The general principle of AO is to measure the aberrations introduced by the media between an object of interest and its image with a wavefront sensor, analyze the measurements, and calculate a correction with a control computer. The corrections are applied to a deformable mirror (DM) positioned in the optical path between the object and its image, thereby ena‐ bling high-resolution imaging of the object.

Modern telescopes with integrated AO systems employ the laser guide star technique [2] to create an artificial reference object above the earth's atmosphere. Analogously, the vast ma‐

jority of present-day vision research AO systems employ a single point source on the retina as a reference object for aberration measurements, consequently termed guide star (GS). AO correction is accomplished with a single DM in a plane conjugated to the pupil plane. An AO system with one GS and one DM will henceforth be referred to as single-conjugate AO (SCAO) system. Aberrations in such a system are measured for a single field angle and cor‐ rection is uniformly applied over the entire field of view (FOV). Since the eye's optical aber‐ rations are dependent on the field angle this will result in a small corrected FOV of approximately 2 degrees [3]. The property of non-uniformity is shared by most optical aber‐ rations such as e.g. the well known primary aberrations of coma, astigmatism, field curva‐ ture and distortion.

of the cornea, the pupil, and the lens, project light from the outside world to the sensory retina (Fig. 1, left). The amount of light that enters the eye is controlled by pupil constric‐ tion and dilation. The human retina is a layered structure approximately 250 µm thick [23, 24], with a variety of neurons arranged in layers and interconnected with synapses

Dual Conjugate Adaptive Optics Prototype for Wide Field High Resolution Retinal Imaging

http://dx.doi.org/10.5772/53640

5

**Figure 1.** Schematic drawings of the eye (left) and the layered retinal structure (right). (Webvision, http://webvi‐

Visual input is transformed in the retina to electrical signals that are transmitted via the optic nerve to the visual cortex in the brain. This process begins with the absorption of photons in the retinal photoreceptors, situated at the back of the retina, which stimulate several interneurons that in turn relay signals to the output neurons, the retinal ganglion cells. The ganglion cell nerve fiber axons exit the eye through the optic nerve head (blind

Unlike the regularly spaced pixels of equal size in a CCD chip the retinal photoreceptor mo‐ saic is an inhomogeneous distribution of cone and rod photoreceptors of various sizes. The central retina is cone-dominated with a cone density peak at the fovea, the most central part of the retina responsible for sharp vision, with a decrease in density towards the rod-domi‐ nated periphery. Cones are used for color and photopic (day) vision and rods are used for

Blood is supplied to the retina through the choroidal and retinal blood vessels. The choroi‐ dal vessels line the outside of the eye and supply nourishment to the photoreceptors and outer retina, while the retinal vessels supply inner retinal layers with blood. Retinal capilla‐ ries, the smallest blood vessels in the eye, branch off from retinal arteries to form an intricate network throughout the whole retina with the exception of the foveal avascular zone (FAZ). The FAZ is the capillary-free region of the fovea that contains the foveal pit where the cones are most densely packed and are completely exposed to incoming light. Capillaries form a superficial layer in the nerve fiber layer, a second layer in the ganglion cell layer, and a third

sion.med.utah.edu/book/part-i-foundations/simple-anatomy-of-the-retina/)

(Fig. 1, right).

spot).

scotopic (night) vision.

layer running deeper into the retina.

A method to deal with this limitation of SCAO was first proposed by Dicke [4] and later de‐ veloped by Beckers [5]. The proposed method is known as multiconjugate AO (MCAO) and uses multiple DMs conjugated to separate turbulent layers of the atmosphere and several GS to increase the corrected FOV. In theory, correcting (in reverse order) for each turbulent lay‐ er could yield diffraction limited performance over the entire FOV. However, as is the case for both the atmosphere and the eye, aberrations do not originate solely from a discrete set of thin layers but from a distributed volume. By measuring aberrations in different angular directions using several GSs and correcting aberrations in several layers of the eye using multiple DMs (at least two), it is possible to correct aberrations over a larger FOV than com‐ pared to SCAO.

The concept of MCAO for astronomy has been the studied extensively [6-12], a number of experimental papers have also been published [13-16], and on-sky experiments have recent‐ ly been launched [17]. However, MCAO for the eye is just emerging, with only a few pub‐ lished theoretical papers [3, 18-21]. Our group recently published the first experimental study [21] and practical application [22] of this technique in the eye, implementing a labora‐ tory demonstrator comprising multiple GSs and two DMs, consequently termed dual-conju‐ gate adaptive optics (DCAO). It enables imaging of retinal features down to a few microns, such as retinal cone photoreceptors and capillaries [22], the smallest blood vessels in the reti‐ na, over an imaging area of approximately 7 x 7 deg2 . It is unique in its ability to acquire single images over a retinal area that is up to 50 times larger than most other research based flood illumination AO instruments, thus potentially allowing for clinical use.

A second-generation Proof-of-Concept (PoC) prototype based on the DCAO laboratory demonstrator is currently under construction and features several improvements. Most sig‐ nificant among those are changing the order in which DM corrections are imposed and the implementation of a novel concept for multiple GS creation (patent pending).


**1. Introduction**

Bacterial survival and persistence in an inappropriate substrate can be defined as the ability of bacteria to tolerate exposure to lethal concentrations of bactericidal antibiotics. This view

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

was first noticed in 1944 regarding that treatment of cultures of *Staphylococcus aureus* with high concentrations of penicillin did not kill all the strains, and that a fraction of the order of 10−6 of these strains survived. Bacteria can cause frequently infections in humans, and the resistance to antibiotics is a primary cause of disease and sometimes death in the intensivecare units of hospitals worldwide [1] and the cause of numerous clinical problems [2]. The development and increase of resistance among pathogens causing nosocomial and community-acquired infections are known to be associated with the widespread utilization of antibiotics [3–5].

The presence of antibiotic resistance strains in environment may be understood as a response to the selective pressures. Thus, any antibiotic use will provide a selective pressure that perpetuates resistant bacteria. The introduction of antibiotics in the treatment of diseases that were prior incurable, enabled the effective treatment of these, promoting the increase in longevity. This kind of medicine is widely used in the treatment of people in the community and health services and is also used to treat animals in agricultural environments. Thus, the increasing levels of resistance are compromising the effectiveness of them. Therefore, it is essential that we assess the use of antibiotics carefully, regardless of setting, and use them only when necessary, to avoid promoting the development of resistance among bacteria [12]. Various infections are caused by important pathogens, such as *Staphylococcus aureus*, and up to 50% are resistant to stronger drugs, such as methicillin [13], and with *E. coli* [12, 14–16] the

Antibiotic Resistance among *Escherichia coli*: Isolates and Novel Approaches to the Control of *E. coli* Infections

http://dx.doi.org/10.5772/67400

101

The food supply can be a source of antimicrobial-resistant and virulent *E. coli* strains that could be lethal to humans or, in a major scale, can cause intestinal as well as extra intestinal infections. *Escherichia coli* (**Figure 1**) is a ubiquitous and versatile microorganism, moving from commensal to opportunistic and specialized virulent bacteria, with potential to cause different diseases. To cause infection, harmless commensal *E. coli* can acquire a set of combination of mobile genetic elements to become a highly adapted pathogen capable of causing different diseases in different hosts, ranging from gastroenteritis to extra intestinal infections of the urinary tract, bloodstream, and central nervous system. Seven diarrheagenic and two

Interestingly, one outbreak happens, caused by one not expected *E. coli* pathotype [18] in 2011. An uncommon high number of haemolytic uremic syndrome (HUS) cases were reported in

extra intestinal *E. coli* pathovars can cause disease in various hosts.

**Figure 1.** *E. coli* in scanning electronic microscopy [17].

resistant trait is growing.

 Various antimicrobial agents that are in use today are antibiotic, antiviral, antifungal, and antiparisitic drugs. An antibiotic is a type of antimicrobial agent produced by a fungi or a bacterium that has a direct influence on other microbes, specifically bacteria. Many of antibiotic resistance genes are found on transposons, integrons, and/or plasmids that can be transferred to another bacteria belonging to the same or different species [6] and resistance elements can be transferred to the human commensal or pathogenic microbiome [7].

The genes conferring resistance to antibiotics have been widely distributed in the environment since before the introduction of antibiotic chemotherapies, but human activities are probably the major driven force of the resistant bacteria found in air and water, principally with *S. aureus* and *E. coli* isolates. The antibiotic presence in the environment can exert selective pressure on the organisms living closely, but the mechanisms of cross resistance to antibiotics are unknown [6].

Studies with animals [8] showed that the proximity with humans has the tendency to generate more antibiotic-resistant enteric bacteria as saw with African baboons. With birds, a study [9] showed that 8% of *E. coli* isolated from arctic region presented resistance. This can enhance the pressure of antibiotic resistance by an anthropogenic activity. In pristine environments, the determinants of an antibiotic resistance existed naturally and were probably subjected to horizontal gene transfer [10]. This predisposition for the genetic exchange of resistance elements has facilitated the antibiotic resistance in pathogenic bacteria.

Antibiotics kill or inhibit bacteria that are susceptible to that antibiotic. Bacteria that are intrinsically resistant or that can acquire resistance will survive and replace the drug-susceptible bacteria. The production of an antibiotic is associated with the presence of genes encoding one or more self-protection processes. Antibiotic biosynthesis gene clusters that encode resistance proteins specific to the compound made (modification of the compound or target) or multifunctional (efflux systems) are important systems. The resistance genes that are contiguous with the biosynthesis genes could be involved in regulation of the biosynthetic pathway.

There are consistent results [11] that showed that antibiotic resistance genes are found in natural sources. Another form of resistance in isolates not producing antibiotics is the mutation of the target gene product, which reduces or prevents inhibition by antibiotic binding. Spontaneous mutations causing resistance often lead to different bacterial phenotypes.

The presence of antibiotic resistance strains in environment may be understood as a response to the selective pressures. Thus, any antibiotic use will provide a selective pressure that perpetuates resistant bacteria. The introduction of antibiotics in the treatment of diseases that were prior incurable, enabled the effective treatment of these, promoting the increase in longevity. This kind of medicine is widely used in the treatment of people in the community and health services and is also used to treat animals in agricultural environments. Thus, the increasing levels of resistance are compromising the effectiveness of them. Therefore, it is essential that we assess the use of antibiotics carefully, regardless of setting, and use them only when necessary, to avoid promoting the development of resistance among bacteria [12].

Various infections are caused by important pathogens, such as *Staphylococcus aureus*, and up to 50% are resistant to stronger drugs, such as methicillin [13], and with *E. coli* [12, 14–16] the resistant trait is growing.

The food supply can be a source of antimicrobial-resistant and virulent *E. coli* strains that could be lethal to humans or, in a major scale, can cause intestinal as well as extra intestinal infections. *Escherichia coli* (**Figure 1**) is a ubiquitous and versatile microorganism, moving from commensal to opportunistic and specialized virulent bacteria, with potential to cause different diseases. To cause infection, harmless commensal *E. coli* can acquire a set of combination of mobile genetic elements to become a highly adapted pathogen capable of causing different diseases in different hosts, ranging from gastroenteritis to extra intestinal infections of the urinary tract, bloodstream, and central nervous system. Seven diarrheagenic and two extra intestinal *E. coli* pathovars can cause disease in various hosts.

Interestingly, one outbreak happens, caused by one not expected *E. coli* pathotype [18] in 2011. An uncommon high number of haemolytic uremic syndrome (HUS) cases were reported in

**Figure 1.** *E. coli* in scanning electronic microscopy [17].

was first noticed in 1944 regarding that treatment of cultures of *Staphylococcus aureus* with high concentrations of penicillin did not kill all the strains, and that a fraction of the order of 10−6 of these strains survived. Bacteria can cause frequently infections in humans, and the resistance to antibiotics is a primary cause of disease and sometimes death in the intensivecare units of hospitals worldwide [1] and the cause of numerous clinical problems [2]. The development and increase of resistance among pathogens causing nosocomial and community-acquired infections are known to be associated with the widespread utilization of anti-

100 *Escherichia coli* Escherichia coli - Recent Advances on Physiology, Pathogenesis and Biotechnological Applications - Recent Advances on Physiology, Pathogenesis and Biotechnological Applications

Antimicrobial is a general term for drugs, chemicals, and/or other substances that either kill or slow the microbial metabolism. Various antimicrobial agents that are in use today are antibiotic, antiviral, antifungal, and antiparisitic drugs. An antibiotic is a type of antimicrobial agent produced by a fungi or a bacterium that has a direct influence on other microbes, specifically bacteria. Many of antibiotic resistance genes are found on transposons, integrons, and/or plasmids that can be transferred to another bacteria belonging to the same or different species [6] and resistance elements can be transferred to the human commensal or pathogenic

The genes conferring resistance to antibiotics have been widely distributed in the environment since before the introduction of antibiotic chemotherapies, but human activities are probably the major driven force of the resistant bacteria found in air and water, principally with *S. aureus* and *E. coli* isolates. The antibiotic presence in the environment can exert selective pressure on the organisms living closely, but the mechanisms of cross resistance to anti-

Studies with animals [8] showed that the proximity with humans has the tendency to generate more antibiotic-resistant enteric bacteria as saw with African baboons. With birds, a study [9] showed that 8% of *E. coli* isolated from arctic region presented resistance. This can enhance the pressure of antibiotic resistance by an anthropogenic activity. In pristine environments, the determinants of an antibiotic resistance existed naturally and were probably subjected to horizontal gene transfer [10]. This predisposition for the genetic exchange of resistance ele-

Antibiotics kill or inhibit bacteria that are susceptible to that antibiotic. Bacteria that are intrinsically resistant or that can acquire resistance will survive and replace the drug-susceptible bacteria. The production of an antibiotic is associated with the presence of genes encoding one or more self-protection processes. Antibiotic biosynthesis gene clusters that encode resistance proteins specific to the compound made (modification of the compound or target) or multifunctional (efflux systems) are important systems. The resistance genes that are contiguous with the biosynthesis genes could be involved in regulation of

There are consistent results [11] that showed that antibiotic resistance genes are found in natural sources. Another form of resistance in isolates not producing antibiotics is the mutation of the target gene product, which reduces or prevents inhibition by antibiotic binding. Spontaneous mutations causing resistance often lead to different bacterial phenotypes.

ments has facilitated the antibiotic resistance in pathogenic bacteria.

biotics [3–5].

microbiome [7].

biotics are unknown [6].

the biosynthetic pathway.

Europe, most precisely in Germany. The implicated agent was an enterohemorrhagic *E. coli* (EHEC), which presented virulence traits of both, a verotoxigenic *E. coli* (VTEC) and enteroaggregative *E. coli* (EAEC).

A relevant study involving enterotoxigenic *E. coli* STb toxin was conducted in 2014 [19], regarding the STb toxigenicity. STb is a heat-resistant toxin responsible for diarrhea in farm animals and in humans as well. The toxic effect of STb in host cells is due to allowing the passage of electrolytes and water through the paracellular space. The authors [19] demonstrated that STb could promote the delocalization of transmembrane proteins such as claudin-1.

This event among others can prove how the versatility of this bacterium can reach a worldwide proportion, with a high concern for public health. These types of outbreaks are not solely a consequence of health conditions in developing countries. With the increase in international travel and trade globalization, diarrheagenic *E. coli* has become a worldwide public health threat [20].

On the other hand, the main characteristic of intracellular bacteria is the ability to survive within the macrophages. In this context, some important pathogens are *L. monocytogenes, Mycobacterium tuberculosis* and, *M. leprae*. Invasion of the macrophage is also a parasite's getaway strategy. Though paradoxical, the last mechanism is benign to the harborer, while the lack of cell penetration by bacteria can induce a strong inflammatory effect and excessive injury to the host.

1. Antibodies

2. Cytokines produced by T cells

http://dx.doi.org/10.5772/67400

103

**I. Natural barriers against infection II. Innate immunity III. Acquired immunity**

Antibiotic Resistance among *Escherichia coli*: Isolates and Novel Approaches to the Control of *E. coli* Infections

protein, complement)

macrophages

1. Integrity of skin and mucosa 1. Extracellular molecules (C reactive

2. Mucociliar movement 2. Natural killer cells, neutrophils,

3. pH variations 3. Chemokines, cytokines – 4. Antimicrobial substances – –

Adaptive immunity, principally by means of antibodies plays an important function versus bacteria outside of the cell. Antibodies may exert its inhibition in three steps: (i) opsonization, (ii) activation of the complementary system, and (iii) furthering the neutralization of bacteria

Extracellular bacteria are prone to undoing when phagocytozed. So subverting this system, they developed substances such as evasive mechanism with an antiphagocytic system.

Antibodies directed against these substances not only avoid their action, also facilitate phagocytosis, while neutrophils and macrophages have receivers for the fc part of immunoglobulin

The antibodies also coassist in the destruction of complement by bacteria, and activate this system by the classical pathway. Through neutralization mechanism, IgA antibodies, in particular, can bind to bacteria and therefore prevent the latter from settling on the intestinal mucosa and the respiratory tract. Antibodies bind frequently to toxins produced from bacteria, such as tetanus (*Clostridium tetani*) and diphtheria toxin (*Corynebacterium diphtheriae*), and neutralize the action of these metabolites. The fine balance between health and disease is found in this scenario, in which deficiency is as much as excess may result in tissue damage.

A mature human gut harbors a vast number of bacterial resident microbiota, accounting for more than 1014 individual bacteria. Notably, the composition of the microbiota is individual host specific and the type of species living in the gastrointestinal tract varies with the host age, diet, habits, health, and idiosyncratic status [22]. The intestinal mucosa is a first contact between the immune system and the external environment and plays a central role in a

or their metabolites.

Note: Ref. [21] with modifications.

**Table 1.** Barriers against infectious microorganisms.

(opsonization).

**3.** *E. coli* **antibiotic resistance**

On the other hand, antibiotic resistance rates in *E. coli* are rapidly rising, especially concerning to the use of fluoroquinolones and third- and fourth-generation cephalosporin. These strains are acquired predominantly in the community [12]. In these conditions, drug-resistant *E. coli* are readily acquired via the consumption of contaminated food and beverage. Some authors [14] studied 287 *E. coli* samples isolated from meats regarding their virulence factors. They observed that drugresistant isolates had similar characteristics to those collected from the same types of meat.

This review shows the immune view of *E. coli*, and focus on the presence of these bacteria highlighting the acquisition of resistance and discussing various aspects of *E. coli* pathotypes. Because the antibiotic treatment is our primary method of threaten diseases, studies on this field are important to a better understanding of bacterial evasion, circumventing, and subverting mechanisms to acquire resistance characteristics.


**Paloma Kiwan1 , Vincent Cicculli1,2, Morena Gasparine1, Remi Charrel2, and Alessandra Falchi1**

1UR7310 BIOSCOPE, Laboratoire de Virologie, Université de Corse-Inserm, 20250 Corte, France 2Unité des Virus Emergents (UVE), Aix-Marseille Université, IRD 190, INSERM 1207, IHU Méditerranée Infection, Marseille, France

Nowadays, emerging pathogens with vector transmission are of major importance. Corsica, a Mediterranean island, is characterized by several factors supporting the spread of vector-borne diseases. Mass tourism and extensive agriculture favor interactions between humans, livestock, and wild fauna. Moreover, Corsica, located on the avian migration route, allows the transport of new invasive tick species. Favored by global warming, ticks succeed in settling and circulating new diseases, including Crimean– Congo hemorrhagic fever (CCHF). CCHF is a common zoonotic viral infection transmitted by ticks. In 2016, human cases were detected in Spain. CCHF was also detected in ticks in Spain and Italy. In Corsica, domestic animals, seropositive for this virus, are detected in cattle (13%) and sheep (2,5%). However, no human clinical cases have been reported to date. Furthermore, CCHF is not detected in ticks circulating in Corsica despite the large circulation of its main vector, Hyalomma marginatum. It is necessary to strengthen the preparation for CCHF emergence. Molecular detection of the CCHF virus in ticks collected from domestic animals will be conducted in parallel with an 18-month epidemiological survey targeting populations at high risk for CCHF infection (farmers, slaughterhouse workers, and their families). A questionnaire will be conducted to assess exposure factors in these populations, and serum samples will be collected to calculate the prevalence of CCHF and compare it to the general population. Investigating the dynamics of an epidemic as a whole through a One Health approach is critical to understand the situation of CCHF in Corsica and closely monitor its possible emergence.

**Raise public health awareness in Corsica and neighboring regions.**

**Evaluate the exposure factors Put in place preventive measurements**

#### Authors

Paloma Kiwan1, Vincent Cicculli1,2, Morena Gasparine1, Remi Charrel2, and Alessandra Falchi 1

1 UR7310 BIOSCOPE, Laboratoire de Virologie, Université de Corse-Inserm, 20250 Corte, France

2 Unité des Virus Emergents (UVE), Aix-Marseille Université, IRD 190, INSERM 1207, IHU Méditerranée Infection, Marseille, France

#### Introduction: Emerging Diseases

Among pathogens responsible for emerging infectious diseases, those with vector transmission are of major importance.

### Crimean-Congo hemorrhagic virus (CCHF)

The most widespread tick-born human disease, due to the extensive geographical distribution of its principal vector Hyalomma marginatum. CCHF is a zoonotic infection, asymptomatic in reservoir animals but responsible for moderate to severe hemorrhagic disease in humans, with a lethal rate reaching 40%.

CCHF antibodies detected in cattle (13%) and sheep ( 2,5%)

CCHF detected No close nairovirus detected

Study objective: Strengthen the preparation for CCHF emergence via a OneHealth approach.

#### Methods

Importance

**Evaluate the source of a possible CCHF epidemic.**


*2.2. Effects of the methanol Extract and Isolated Lignans on LPS-Induced PGE2 and TNF-α Secretion*

Lipopolysaccharide (LPS), known as a major factor involved in inflammation, is a component of the outer membrane of Gram-negative bacteria. Moreover, it is called endotoxin because of its endotoxic properties. LPS contributes to the pathogenicity of bacteria and can activate several immune cells, including macrophages [20]. In this study, LPS was used for inducing inflammation in RAW 264.7 macrophages. Incubation of the cells with LPS (5 μg/mL) for 24 h markedly increased PGE2 and TNF-α secretion (Figure 3). The methanol extract of *M. sirikitiae* leaves was first assessed for the inhibitory effects on LPS-induced PGE2 and TNF-α secretions. Treatment of the cells with the methanol extract of *M. sirikitiae* leaves resulted in a decrease in LPS-induced PGE2 secretion in a dosedependent manner, and the maximal inhibitory effect of the methanol extract was observed at a concentration of 10 μg/mL (Figure 3A). However, the methanol extract exhibited little effect on inhibition of LPS-induced TNF-α secretion at the same concentration and did not inhibit LPS-induced TNF-α secretion at a concentration of 1 μg/mL (Figure 3B). These results indicated that the compounds found in *M. sirikitiae* leaves might have antiinflammatory effects by reducing the secretion of PGE2 in RAW 264.7 macrophages.

**Figure 3.** Effects of the methanol extract of *M. sirikitiae* leaves on LPS-induced PGE2 and TNF-α secretion in RAW 264.7 cells; Serum-starved cells were pretreated with various concentrations of 0, 1, and 10 μg/mL of crude methanol extract for 3 h, and then stimulated with LPS (5 μg/mL) for 24 h at 37 ◦C. The levels of PGE2 and TNF-α secreted into the medium were assessed by ELISA assay. The PGE2 (**A**) and TNF-α (**B**) levels were quantified using a standard curve and expressed as the mean ± SEM (*n* = 3). \*, *p* < 0.05 vs. vehicle; #, *p* < 0.05 vs. LPS.

Lignans are polyphenols, and the main components of *M. sirikitiae* leaves. Our previous study has revealed that lignans (−)-epieudesmin (**1**), (−)-phylligenin (**2**), 2-(3,4 dimethoxyphenyl)-6-(3,5-dimethoxyphenyl)-3,7-dioxabicyclo[3.3.0]octane (**3**), magnone A (**4**), mitrephoran (**5**), and 3 ,4-*O*-dimethylcedrusin (**6**) are the main secondary metabolites in *M. sirikitiae* leaves [10]. As shown in Figure 4A, treatment of the cells with (−)-phylligenin (**2**) (10 μg/mL) significantly inhibited LPS-induced PGE2 secretion. However, (−)-epieudesmin (**1**), 2-(3,4-dimethoxyphenyl)-6-(3,5-dimethoxyphenyl)-3,7 dioxabicyclo[3.3.0]octane (**3**), magnone A (**4**), mitrephoran (**5**), and 3 ,4-*O*-dimethylcedrusin (**6**) had no effect on the inhibition of LPS-induced PGE2 secretion. These results are consistent with previous studies, which reported that phylligenin and koreanaside A, lignans isolated from *Forsythia koreana* fruits and flowers, could inhibit PGE2 secretion in LPS-stimulated RAW 264.7 cells in a dose-dependent manner [15,16]. Moreover, the antiinflammation of koreanaside A was also represented by the inhibitory effects on NO, TNF-α, and IL-6 productions [16].

**Figure 4.** Effects of the methanol extract and isolated lignans **1**–**6** from *M. sirikitiae* leaves on LPSinduced PGE2 and TNF-α secretion in RAW 264.7 cells; Serum-starved cells were pretreated with crude methanol extract (C) (10 μg/mL), lignans (5 or 10 μg/mL), or indomethacin (10 μM) for 3 h, and then stimulated with LPS (5 μg/mL) for 24 h at 37 ◦C. The levels of PGE2 and TNF-α secreted into the medium were assessed by ELISA assay. The relative PGE2 (**A**) and TNF-α (**B**) levels were quantified using a standard curve and expressed as the mean ± SEM (*n* = 3). \*, *p* < 0.05 vs. vehicle; #, *p* < 0.05 vs. LPS.

Interestingly, all lignans isolated from the caulis of *Urceola rosea* exhibited antiinflammatory activities by suppressing LPS-induced synthesis of NO, TNF-α, and/or IL-6 in RAW 264.7 cells. In addition, among those lignans, ecdysanol A and ecdysanol F exhibited potent anti-inflammatory activity against TNF-α secretion with IC50 values of 22.9 and 41.9 μM, respectively [17]. Consistent with this previous study, our results showed that among those six lignans isolated from *M. sirikitiae* leaves, 2-(3,4-dimethoxyphenyl)-6- (3,5-dimethoxyphenyl)-3,7-dioxabicyclo[3.3.0]octane (**3**) and mitrephoran (**5**) (5 μg/mL) significantly inhibited LPS-induced TNF-α secretion in RAW 264.7 cells (Figure 4B).


**3.3. Novel mechanisms of the wobble (w)**↔**Watson-Crick (WC) tautomeric interconversions in the canonical and incorrect DNA base pairs as a key to understand origins of spontaneous transitions and transversions**

For the first time, a novel theoretical approach to elucidate microstructural mechanisms of incorporation and replication point errors arising at the DNA replication was proposed. We show for the first time that pairs of nucleotide bases with Watson-Crick architecture of the H-bonding—classical, long, short, in which one or both bases are in the main or rare tautomeric forms, are in a slow tautomeric equilibrium with the corresponding wobble base pairs in comparison with the time, in which high-fidelity DNA polymerase spends on the incorporation of one nucleotide into the DNA double helix (~8.3 × 10−4 s [67]). In fact, a novel pathway of the chemical reaction was discovered—tautomerization with significant changes of the geometry of the base pair—from Watson-Crick to wobble and *vice versa*.

We have discovered novel structural hypostases of the classical A·T(WC) and G·C(WC) Watson-Crick DNA base pairs arising due to their ability to switch into the wobble А\*·Т↑(w), А·Т\*O2↑(w), А·Т\*↓(w) and G·C\*↑(w), G\*·C↓(w), G·C\*↓(w), G\*·C↑(w) H-bonded mismatches containing rare tautomers (**Figure 2**) [68]. Estimated populations of the tautomerized states of the А·Т(WC) (6.1 × 10−9–1.5 × 10−7) and G·C(WC) (4.2 × 10−11–1.4 × 10−9) base pairs in the continuum with ε = 4 correspond to the interface of the protein-nucleic acid interactions. This evidences their involvement in nucleation of spontaneous point replication errors in DNA arising with frequencies ~10−11–10−9 errors *per* replicated nucleotide.

We found for the first time the intrinsic ability of the purine·pyrimidine (A·C [69, 70] and G·T [69, 71]), purine·purine (A·A [72], G·G [72] and A·G [73]) and pyrimidine·pyrimidine (С·С [74], Т·Т [74] and С·T [73]) DNA base mispairs to perform wobble↔Watson-Crick tautomeric transitions *via* the sequential intrapair DPT and subsequent shifting of the bases relative to each other (**Figure 3**, **Table 2**). These nondissociative tautomerizations *via* the sequential PT are controlled by the highly stable (ΔEint > 100 kcal·mol−1), highly polar and zwitterionic transition states of the type (protonated base)·(deprotonated base). These interconversions are accompanied by a significant rebuilding of the base mispairs with Watson-Crick architecture into the mismatches wobbled toward both DNA minor and major grooves and *vice versa*.

**Figure 2.** Energetic profiles of the mutagenic tautomerization *via* the wobbling of the (**a**) A·T(WC) and (**b**) G·C(WC) DNA base pairs to the H-bonded mismatches containing rare tautomers (B3LYP/6–311++G(d,p) level of theory, ε = 1) [68].

Notably, each of the discussed tautomerizations is realized precisely through four different topological and energetic pathways. The number of mobile protons (two in each pair) and number of wobbling directions of WC base pairs (two by the number of the grooves in DNA minor and major) determines the number of tautomerization pathways. Characteristically, in

**Figure 3.** Energetic profiles and stationary structures on the potential energy hypersurface of the biologically important transformations *via* the PT, accompanied by the shifting of the bases relative to each other within a base pair into the sides of the DNA minor or major grooves, leading to the occurrence of the spontaneous transitions and transversions—

Renaissance of the Tautomeric Hypothesis of the Spontaneous Point Mutations in DNA: New Ideas…

http://dx.doi.org/10.5772/intechopen.77366

37

incorporation and replication errors (B3LYP/6–311++G(d,p) level theory, ε = 1).

Renaissance of the Tautomeric Hypothesis of the Spontaneous Point Mutations in DNA: New Ideas… http://dx.doi.org/10.5772/intechopen.77366 37

**3.3. Novel mechanisms of the wobble (w)**↔**Watson-Crick (WC) tautomeric interconversions in the canonical and incorrect DNA base pairs as a key to**

of the geometry of the base pair—from Watson-Crick to wobble and *vice versa*.

arising with frequencies ~10−11–10−9 errors *per* replicated nucleotide.

For the first time, a novel theoretical approach to elucidate microstructural mechanisms of incorporation and replication point errors arising at the DNA replication was proposed. We show for the first time that pairs of nucleotide bases with Watson-Crick architecture of the H-bonding—classical, long, short, in which one or both bases are in the main or rare tautomeric forms, are in a slow tautomeric equilibrium with the corresponding wobble base pairs in comparison with the time, in which high-fidelity DNA polymerase spends on the incorporation of one nucleotide into the DNA double helix (~8.3 × 10−4 s [67]). In fact, a novel pathway of the chemical reaction was discovered—tautomerization with significant changes

We have discovered novel structural hypostases of the classical A·T(WC) and G·C(WC) Watson-Crick DNA base pairs arising due to their ability to switch into the wobble А\*·Т↑(w), А·Т\*O2↑(w), А·Т\*↓(w) and G·C\*↑(w), G\*·C↓(w), G·C\*↓(w), G\*·C↑(w) H-bonded mismatches containing rare tautomers (**Figure 2**) [68]. Estimated populations of the tautomerized states of the А·Т(WC) (6.1 × 10−9–1.5 × 10−7) and G·C(WC) (4.2 × 10−11–1.4 × 10−9) base pairs in the continuum with ε = 4 correspond to the interface of the protein-nucleic acid interactions. This evidences their involvement in nucleation of spontaneous point replication errors in DNA

We found for the first time the intrinsic ability of the purine·pyrimidine (A·C [69, 70] and G·T [69, 71]), purine·purine (A·A [72], G·G [72] and A·G [73]) and pyrimidine·pyrimidine (С·С [74], Т·Т [74] and С·T [73]) DNA base mispairs to perform wobble↔Watson-Crick tautomeric transitions *via* the sequential intrapair DPT and subsequent shifting of the bases relative to each other (**Figure 3**, **Table 2**). These nondissociative tautomerizations *via* the sequential PT are controlled by the highly stable (ΔEint > 100 kcal·mol−1), highly polar and zwitterionic transition states of the type (protonated base)·(deprotonated base). These interconversions are accompanied by a significant rebuilding of the base mispairs with Watson-Crick architecture into the mismatches wobbled toward both DNA minor and major grooves and *vice versa*.

**Figure 2.** Energetic profiles of the mutagenic tautomerization *via* the wobbling of the (**a**) A·T(WC) and (**b**) G·C(WC) DNA base pairs to the H-bonded mismatches containing rare tautomers (B3LYP/6–311++G(d,p) level of theory, ε = 1) [68].

**understand origins of spontaneous transitions and transversions**

36 Mitochondrial DNA - New Insights

**Figure 3.** Energetic profiles and stationary structures on the potential energy hypersurface of the biologically important transformations *via* the PT, accompanied by the shifting of the bases relative to each other within a base pair into the sides of the DNA minor or major grooves, leading to the occurrence of the spontaneous transitions and transversions incorporation and replication errors (B3LYP/6–311++G(d,p) level theory, ε = 1).

Notably, each of the discussed tautomerizations is realized precisely through four different topological and energetic pathways. The number of mobile protons (two in each pair) and number of wobbling directions of WC base pairs (two by the number of the grooves in DNA minor and major) determines the number of tautomerization pathways. Characteristically, in

Mutagenic pressure of the analogues of DNA bases could be explained within the framework of the proposed model of the w↔WC mutagenic tautomerization. In particular, mutagenic action of the analogue of C-6H,8H-3,4-dihydropyrimido[4,5-c] [1, 2]oxazin-7-one [11, 12] increases the population of the G·P\*↑ (4.5 × 10−3) and G·P\*↓ (1.4 × 10−4) base mispairs by its participation in comparison with the analogical values for the canonical C DNA base. Mutagenic activity of the halogen derivatives of the uracil base is associated with the decreas-

induced mutations (35 [71]), which is in good accordance with experimental data (from 20

All long purine·purine DNA base mispairs can acquire enzymatically competent conformations—А\*·Аsyn(TF), G·Аsyn, A\*·G\*syn and G·G\*syn—through the A\*·A(WC)↔A\*·Asyn(TF), G·A(WC)↔G·Asyn, A\*·G\*(WC)↔A\*·G\*syn and G·G\*(WC)↔G·G\*syn conformational transitions [77], eventually guaranteeing their chemical incorporation into the newly synthesized structure of the DNA double helix (TF-Topal-Fresco nucleobase pair [10]; *syn*-*syn*-orientation of the base according the sugar-phosphate moiety) (**Figure 4**). Characteristic time of these nondissociative conformational transitions (~10−7 s) is much less than the period of time the high-fidelity DNA polymerase spends on incorporating one nucleotide into the DNA double helix (~8.3 × 10−4 s [67]). So-called long A\*·A(WC), G·A(WC), A\*·G\*(WC) and G·G\*(WC) DNA base mispairs have been outlined as "node stations" on the way of the formation of the enzymatically competent conformations arising in the recognition pocket of the high-fidelity DNA

**3.5. Physico-chemical scenarios of the origin of the replication and incorporation**

for three other cases, when G\*, T\* and C\* belong to the template strand of DNA.

In the framework of such qualitatively new model conceptions, we were able to shed light on the microstructural mechanisms of the occurrence of point mutations—replication and

Thus, the spontaneous mutagenic tautomerization of the Watson-Crick pairs of nucleotide bases into the wobble base mispairs, which includes the A\*, T\*, G\* and C\* mutagenic tautomers, has been established to be the source of the generation of the mutagenic tautomers of the DNA bases arising at the separation of DNA strands. At this juncture, *replication errors* would arise in the following way (as an example, we would consider the case, when A\* belongs to the template strand of DNA): A\* + C → A\*·C → A·C\*, A\* + A → A\*·A → A\*·Asyn, A\* + G → A \*·G → A·G → A\*·G\* → A\*·G\*syn. Similar schemes of structural transformations, which occur directly in the recognition pocket of the high-fidelity DNA polymerase, would take place also

*Incorporation errors* would occur according to the following scenario: in the recognition pocket of the high-fidelity DNA polymerase, it would form the appropriate wobble base mispair

XU\*(WC) mispairs with Watson-Crick geometry, thus inducing higher frequency

Renaissance of the Tautomeric Hypothesis of the Spontaneous Point Mutations in DNA: New Ideas…

XU(w) (X = H, CH3

, Br, Cl, F) mispairs

39

BrU-calculated frequency of the

http://dx.doi.org/10.5772/intechopen.77366

ing of the transformation barriers of the wobble G·5

of the transitions. The maximal effect is observed for the 5

polymerase at its transition from the open to closed state.

**3.4.** *Anti*↔*syn* **conformational transitions of the long purine-purine DNA**

into the G·5

[75] to 29 [76]).

**mismatches**

**point errors in DNA**

incorporation point errors.

Note: for designations see **Table 1**.

a The time necessary to reach 99.9% of the equilibrium concentration between the reactant and the product of the tautomerization reaction, s.

b Populations of the wobble mispairs containing mutagenic tautomers.

**Table 2.** Energetic and kinetic characteristics of the tautomeric transformations of the classical Watson-Crick or wobble DNA base pairs, which are involved into the processes of the spontaneous point mutagenesis, *via* the DPT accompanied by the substantial changes of their geometry in the continuum with ε = 1.

each case mostly, one pathway is most probable at the origin of the spontaneous point mutations (**Figures 2** and **3**, **Table 2**).

Obtained results are crucial for understanding the microstructural mechanisms of spontaneous transitions and transversions, since they allow us to explain how incorrect purine·pyrimidine, purine·purine and pyrimidine·pyrimidine wobble pairs adapt to the enzymatically competent sizes in the recognition pocket of the high-fidelity DNA polymerase. In particular, established A·C(w) →A·C\*(WC) [70] and G·T(w) → G\*·T(WC) [71] transformations *via* the sequential PT allow us to interpret the X-ray [14, 15] and molecular dynamics simulations data [19] according the acquisition by the wobble A·C(w)/G·T(w) mispairs of the Watson-Crick geometry by their transformation to the A·C\*(WC)/G\*·T(WC) Watson-Crick-like base mispairs by the participation of the C\* and G\* mutagenic tautomers in the recognition pocket of the high-fidelity DNA polymerase. Moreover, we theoretically predicted the G·T(w) → G\*·T(WC) transformation for the wobble G·T(w) base mispair, which was confirmed by an NMR experiment of a DNA duplex [16–18].

Mutagenic pressure of the analogues of DNA bases could be explained within the framework of the proposed model of the w↔WC mutagenic tautomerization. In particular, mutagenic action of the analogue of C-6H,8H-3,4-dihydropyrimido[4,5-c] [1, 2]oxazin-7-one [11, 12] increases the population of the G·P\*↑ (4.5 × 10−3) and G·P\*↓ (1.4 × 10−4) base mispairs by its participation in comparison with the analogical values for the canonical C DNA base. Mutagenic activity of the halogen derivatives of the uracil base is associated with the decreasing of the transformation barriers of the wobble G·5 XU(w) (X = H, CH3 , Br, Cl, F) mispairs into the G·5 XU\*(WC) mispairs with Watson-Crick geometry, thus inducing higher frequency of the transitions. The maximal effect is observed for the 5 BrU-calculated frequency of the induced mutations (35 [71]), which is in good accordance with experimental data (from 20 [75] to 29 [76]).


*Edited by Leszek A. Dobrzański*

The book *Biomaterials in Regenerative Medicine* is addressed to the engineers and mainly medical practitioners as well as scientists and PhD degree students. The book indicates the progress in research and in the implementation of the ever-new biomaterials for the application of the advanced types of prosthesis, implants, scaffolds and implantscaffolds including personalised ones. The book presents a theoretical approach to the synergy of technical, biological and medical sciences concerning materials and technologies used for medical and dental implantable devices and on metallic biomaterials. The essential contents of the book are 16 case studies provided in each of the chapters, comprehensively describing the authors' accomplishments of numerous teams from different countries across the world in advanced research areas relating to the biomaterials applied in regenerative medicine and dentistry. The detailed information collected in the book, mainly deriving from own and original research and R&D works pursued by the authors, will be beneficial for the readers to develop their knowledge and harmonise specific information concerning these topics.

Photo by photo5963 / iStock


*4.3. GOX Activity Measurements*

Enzyme activities were measured using 5 μg of purified recombinant GOX in 50 mM Tris-HCl, 0.1 mM FMN, pH 8.0 and different glycolate (0.05 to 10 mM), L-lactate (0.3 to 10 mM) and 2-hydroxy-octanoate (0.15 to 5 mM) concentrations by an enzyme-coupled reaction at 30 ◦C. H2O2 produced by GOX activity was quantified in the presence of 0.4 mM *o*-dianisidine and 2 U horseradish peroxidase by measuring the ΔA440nm using a Varian Cary 50 spectrophotometer. KM and kcat values were calculated using SigmaPlot 13.0 software, based on the curve fitting Michaelis–Menten equation: v0 = Vmax[S]/(KM + [S]).