**Approach to the Management of the Pregnant Inflammatory Bowel Disease Patient: Successful Outcome**

Flavio M. Habal *University Health Network University Of Toronto Canada*

## **1. Introduction**

176 Inflammatory Bowel Disease – Advances in Pathogenesis and Management

[6] Seifart, H.I.; Kruger, P.B.; Parkin, D.P; Van Jaarsveld, P.P.; Donald, P.R., J. Chromatogr.,

[9] Cummins, C. L.; O' Neil, W.; Soo, E. C.; Lloyd, D. K.; Wainer I.W. Newspaper

[12] Mocak, J.; Bond, A. M.; Mitchell, S.; Scollary, G. *Pure* & *Appl. Chern.,* 1997, 69, 297-328. [13] Gennaro, M.C.; Calvino, R.; Abrigo, C. Journal of Chromatography B., 2001, 754, 477–

[7] Hansen Jr. E.B.; Dooley, K.L.; Thompson Jr. H.C. J. Chromatogr., 1995, 670, 259. [8] Karlaganis, G.; Peretti, E.; Lauterburg, B.H.; J. Chromatogr., 1987, 420,171.

chromatography B., 1997, 697, 283-288. [10] Song, M.; Xia, B.; Li J. Postgrad Med J., 2006, 82, 130–135.

[11] Vernia, P.; Cittadini, M. ; Caprilli, R. Dig Dis Sci., 1995, 40,305–307.

1993, 619,285.

486.

Inflammatory bowel disease (IBD) encompasses Crohn's disease (CD) and ulcerative colitis (UC) and it affect young adults in their reproductive years (Andres & Friedman, 1999).

Approximately 25% of these patients will conceive after the diagnosis is made. Many women with IBD raise concerns related to the effects of both the disease and medical therapy on fertility, pregnancy and foetal outcomes. On the basis of prospective and registry data, it is known that women with IBD have a higher risk of low-birth-weight deliveries and pre-term deliveries (Cornish J, 2007). With the exception of patients who have had previous pelvic surgery, the majority of these patients are able to conceive, have a normal pregnancy and a good foetal outcome. Maintaining remission, with drug therapy, prior to conception and during pregnancy is of prime importance. Control of disease activity before conception and during pregnancy is critical to optimize both maternal and fetal health (Mountfield, 2010). Normal maternal weight gain during pregnancy appears to protect against adverse outcome. The majority of drugs used in IBD appear to be safe and their benefit appears to outweigh the risk of disease exacerbation and poor foetal outcome. The newer biologic anti TNF alpha drugs appear to be safe during pregnancy.

Although there are reports of some traces of drugs in breast milk in women taking medications no major undue complication has been reported.

Treatment of IBD patients who are contemplating conception should be tailored to each patient, taking into accounts the patient wishes and concerns. The patient should be followed by a multidisciplinary team including the family physician, the gastroenterologist and the obstetrician.

#### **Preconception advice and counselling**

Preconception care aims to ensure the optimal physical and mental well-being of women and their partners at the onset of and during early pregnancy, to increase the likelihood of normal pregnancy and the delivery of a healthy infant. Pre pregnancy counselling should be an integral part of IBD consultation. Patient understanding of the disease and its changes during pregnancy and breastfeeding is vital for a successful outcome. In a recent

Approach to the Management of the Pregnant

**2. Folic acid, calcium and vitamin D** 

calcium and vitamin D supplementation to prevent bone loss.

as Foetal Alcohol Spectrum Disorders (FASDs) (Davies & Bledsoe, 2005).

improves CD and aggravates ulcerative colitis **(**Lakatos P Let al, 2007).

however, that the risk is decreased in breastfed infants (Yang H et al 1983).

(Caruso, 1998).

**3. Alcohol** 

**4. Smoking** 

**5. Heredity** 

prior to, during and after pregnancy.

ulcerative colitis (UC) (Thompson N et al. 1996).

Inflammatory Bowel Disease Patient: Successful Outcome 179

TPN proved to be helpful and lifesaving in malnourished pregnant women and promoted foetal growth, as shown by the longitudinal ultrasonographic evaluations

Folic acid supplementation is recommended for all pregnant women. Women with IBD may have folic acid deficiency or be taking medications that interfere with folic acid metabolism such as Sulfasalazine **(**Alstead, 2002). Certain patients with Cohn's disease are on low residue diet and may be folate deficient. Thus, pregnant women with IBD should be encouraged to take 5 mg of folic acid per day instead of 1 mg/d as recommended for the general population. Also, patients with IBD on steroid therapy should be encouraged to take

Heavy drinking throughout pregnancy can result in what is known as foetal alcohol syndrome. Patients contemplating pregnancy should be educated about the adverse effects of alcohol. Foetal Alcohol Syndrome (FAS) is a pattern of mental and physical defects clinically defined by growth deficiency, central nervous system damage and dysfunction, and a unique cluster of facial abnormalities. on foetal development. Prenatal alcohol exposure can also cause less pronounced mental, learning and behavioural disabilities in the child, commonly termed

Smoking increases the risk of developing CD and worsens its course, increasing the need for steroids, immunosuppressants and re-operations. It appears that smoking cessation

Tobacco smoking during pregnancy has been associated with placenta previa, placental abruption, premature rupture of membranes (Shah & Bracken, 2000; Mercer, 2008). It can result in small foetal size at 10-19 weeks and may result in early premature birth, low birth weight, and poor foetal outcome. Smoking cessation should be encouraged in the patient

Genetic factors appear to play a role in the pathogenesis of Crohn's disease (CD) and

In association with environmental triggers, heredity increases the risk of developing IBD; however, studies of twins also indicate the involvement of multiple factors (Orholm M, 2000; Tysk C et al 1988). Offspring of patients with IBD are two to 13 times more likely to develop IBD than the general population. The risk of an infant developing IBD is 8% to 11% if one parent has IBD and 20% to 35% if both parents have the disease. Data suggest,

In the general population, approximately 4% of babies born every year have congenital abnormalities, an outcome similar to what is seen in retrospective studies of women whose IBD is treated during their pregnancy. Studies that report prematurity and small-for-

abstract from England the authors showed that there was a lack of knowledge and understanding in the women of reproductive age about their disease, medication and effects of these on pregnancy. It is important to discuss the issues related to pregnancy and conception either at diagnosis or when medication is initially prescribed. In our experience some pregnant women tend to stop their medication in fear of the side effect of the drugs on the developing foetus, with an inherent risk of disease flare up. A questionnaire study by Mountfield et al found that a large proportion of subjects (84%) reported concerns that IBD medications would harm their pregnancy, whereas only 19% women reported concerns about the effect of active IBD on pregnancy. The author reported that patients with active disease had higher risks of adverse outcome with a better outcome if they are in remission (Mountfield, 2010).

It is estimated that more than 50% of pregnancies are not planned hence as soon as the diagnosis is made this issue should be brought up. See table 1, for general pre-pregnancy recommendations.

Folic acid is an important vitamin that should be taken prior to conception since neural tube defect occurs as soon as conception is discovered. It is interesting in the British abstract only 43 women (65.15%) knew the beneficial effect of folic acid pre-conception and during the first trimester of pregnancy (Chakrabarty & Poullis, 2011).

The patient, her partner and her physician should discuss the possibility of disease exacerbation during pregnancy while off treatment and the necessary courses of action in such an event. Treatment choices depend on individual preference, disease severity and potential for drug toxicity. Risks and benefits of maintenance therapies during pregnancy with the best available evidence should be addressed. In addition, breastfeeding should be presented as a favourable option since it confers numerous benefits to both mother and child. The likelihood of medication secretion in breast milk and impact on foetal wellbeing should be approached and encouraged.


Table 1.

#### **Dietary supplementation and nutritional therapy**

**Nutritional therapy:** The average weight gain during pregnancy is 11-16 kg. Early nutritional intervention is indicated in pregnant women with active IBD who may not be gaining weight. Enteral feeding has been anecdotally shown to be associated with normal pregnancy outcome (Teahon, 1991) and total parenteral nutrition (TPN) may be required in very sick IBD patients.

TPN proved to be helpful and lifesaving in malnourished pregnant women and promoted foetal growth, as shown by the longitudinal ultrasonographic evaluations (Caruso, 1998).

## **2. Folic acid, calcium and vitamin D**

Folic acid supplementation is recommended for all pregnant women. Women with IBD may have folic acid deficiency or be taking medications that interfere with folic acid metabolism such as Sulfasalazine **(**Alstead, 2002). Certain patients with Cohn's disease are on low residue diet and may be folate deficient. Thus, pregnant women with IBD should be encouraged to take 5 mg of folic acid per day instead of 1 mg/d as recommended for the general population. Also, patients with IBD on steroid therapy should be encouraged to take calcium and vitamin D supplementation to prevent bone loss.

## **3. Alcohol**

178 Inflammatory Bowel Disease – Advances in Pathogenesis and Management

abstract from England the authors showed that there was a lack of knowledge and understanding in the women of reproductive age about their disease, medication and effects of these on pregnancy. It is important to discuss the issues related to pregnancy and conception either at diagnosis or when medication is initially prescribed. In our experience some pregnant women tend to stop their medication in fear of the side effect of the drugs on the developing foetus, with an inherent risk of disease flare up. A questionnaire study by Mountfield et al found that a large proportion of subjects (84%) reported concerns that IBD medications would harm their pregnancy, whereas only 19% women reported concerns about the effect of active IBD on pregnancy. The author reported that patients with active disease had higher risks of adverse outcome with a

It is estimated that more than 50% of pregnancies are not planned hence as soon as the diagnosis is made this issue should be brought up. See table 1, for general pre-pregnancy

Folic acid is an important vitamin that should be taken prior to conception since neural tube defect occurs as soon as conception is discovered. It is interesting in the British abstract only 43 women (65.15%) knew the beneficial effect of folic acid pre-conception and during the

The patient, her partner and her physician should discuss the possibility of disease exacerbation during pregnancy while off treatment and the necessary courses of action in such an event. Treatment choices depend on individual preference, disease severity and potential for drug toxicity. Risks and benefits of maintenance therapies during pregnancy with the best available evidence should be addressed. In addition, breastfeeding should be presented as a favourable option since it confers numerous benefits to both mother and child. The likelihood of medication secretion in breast milk and impact on foetal wellbeing

**Nutritional therapy:** The average weight gain during pregnancy is 11-16 kg. Early nutritional intervention is indicated in pregnant women with active IBD who may not be gaining weight. Enteral feeding has been anecdotally shown to be associated with normal pregnancy outcome (Teahon, 1991) and total parenteral nutrition (TPN) may be required in

1. Discuss potential pregnancy outcome, breast feeding, and drug therapy

better outcome if they are in remission (Mountfield, 2010).

first trimester of pregnancy (Chakrabarty & Poullis, 2011).

should be approached and encouraged.

General advice pre pregnancy

3. Folic acid supplementation 5 mg daily

5. Establish a record of vaccination and update

**Dietary supplementation and nutritional therapy** 

2. Diet and nutrition

very sick IBD patients.

4. Decrease alcohol intake

3. Stop smoking

Table 1.

recommendations.

Heavy drinking throughout pregnancy can result in what is known as foetal alcohol syndrome. Patients contemplating pregnancy should be educated about the adverse effects of alcohol. Foetal Alcohol Syndrome (FAS) is a pattern of mental and physical defects clinically defined by growth deficiency, central nervous system damage and dysfunction, and a unique cluster of facial abnormalities. on foetal development. Prenatal alcohol exposure can also cause less pronounced mental, learning and behavioural disabilities in the child, commonly termed as Foetal Alcohol Spectrum Disorders (FASDs) (Davies & Bledsoe, 2005).

## **4. Smoking**

Smoking increases the risk of developing CD and worsens its course, increasing the need for steroids, immunosuppressants and re-operations. It appears that smoking cessation improves CD and aggravates ulcerative colitis **(**Lakatos P Let al, 2007).

Tobacco smoking during pregnancy has been associated with placenta previa, placental abruption, premature rupture of membranes (Shah & Bracken, 2000; Mercer, 2008). It can result in small foetal size at 10-19 weeks and may result in early premature birth, low birth weight, and poor foetal outcome. Smoking cessation should be encouraged in the patient prior to, during and after pregnancy.

## **5. Heredity**

Genetic factors appear to play a role in the pathogenesis of Crohn's disease (CD) and ulcerative colitis (UC) (Thompson N et al. 1996).

In association with environmental triggers, heredity increases the risk of developing IBD; however, studies of twins also indicate the involvement of multiple factors (Orholm M, 2000; Tysk C et al 1988). Offspring of patients with IBD are two to 13 times more likely to develop IBD than the general population. The risk of an infant developing IBD is 8% to 11% if one parent has IBD and 20% to 35% if both parents have the disease. Data suggest, however, that the risk is decreased in breastfed infants (Yang H et al 1983).

In the general population, approximately 4% of babies born every year have congenital abnormalities, an outcome similar to what is seen in retrospective studies of women whose IBD is treated during their pregnancy. Studies that report prematurity and small-for-

Approach to the Management of the Pregnant

statistically significant (Raatikainen et al 2011).

UC and CD separately (Mahadevan et al 2007).

aggressively as women who are not pregnant.

making **(**Cornish et al 2007).

Inflammatory Bowel Disease Patient: Successful Outcome 181

incidence of adverse outcomes makes statistical precision difficult; and disease activity was not reported in relation to outcomes. In addition, increased risks of congenital abnormalities associated with 5-ASA, azathioprine and anti-TNF-alpha medications seen in a pooled analysis may be associated with the disease and not the medications. The authors also pointed out the need for a definitive study to determine optimal management and to support the development of new guidelines to assist patients and clinicians in decision-

In a recent publication **by** Raatikainen there was a lower birth weight of newborns from mothers with UC as compared to the general population ( 3317 \_ s.d. 658 g vs. 3506 \_ s.d. 613 g, P = 0.003;3340 \_ s.d. 631 g vs. 3507 \_ s.d. 613 g, P = 0.002) respectively. Newborns in the CD group were also smaller than those in the reference group but the difference was not

A Taiwanese, Asian population database from 2001-2003 study found an increase of preterm births (11.73 vs. 6.25%; p = 0.004) and LBW (12.76 vs. 5.55%; p < 0.001), controlling for maternal characteristics including age, parity and education level (Lin, 2010). In a cohort study from Northern California Kaiser population, pregnant women with IBD were more likely to have a spontaneous abortion (OR: 1.65; 95% CI: 1.09–2.48); an adverse pregnancy outcome (stillbirth, preterm birth or SGA infant; OR: 1.54; 95% CI: 1.00–2.38); or a complication of labour (OR: 1.78; 95% CI: 1.13–2.81). The study did not find a difference in the rate of congenital malformations in control versus IBD patients, either as a group or for

In a review in 1998 Subahani et al found that CD, especially active disease, is associated with decreased birth weights, preterm delivery and caesarean section **(**Subahani & Hamilton, 1998)*.* In a case-control study published by Bush comparing 116 pregnancies in patients with IBD with 56,398 controls, there were no differences in ante partum complications, including chronic hypertension, hyperemesis gravidarum, preterm labour or pre-eclampsia. Among patients with IBD, however, induction of labour (32% versus 24%; P=0.002), chorioamnionitis (7% versus 3%; P=0.04), and caesarean section (32% versus 22%; P=0.007) were all more frequent. Neonatal complications, including low birth weight (LBW), very low birth weight, intrauterine growth restriction, Apgar scores and congenital anomalies, were similar in both groups. Subgroup analysis found a decreased risk of LBW associated with previous IBD

The most recent and only prospective study has just been published by the ECCO-Epicom study (Bertoli et al, 2011)**.** This study from Europe was prospective case-controlled with 332 patients with IBD both Crohn's disease and Ulcerative colitis who were pregnant and were compared to non-IBD pregnant women in the general population. In this study there was no difference in live births, spontaneous or therapeutic abortions, infant death in utero, preterm deliveries and caesarean sections. There was no difference in congenital anomalies or birth weight as compared to outcome in pregnancy in the general population. The only risk that was demonstrated to be associated with congenital anomaly and preterm delivery was older age ≥ 35 years. Smoking was found to be a factor which increased the risk of preterm delivery. Dejaco performed a prospective study assessing risk factors for poor pregnancy outcome in 58 patients with IBD (Dejaco, et al 2006). The authors found that active disease during pregnancy represents a significant risk factor for unfavourable birth outcome. These results support the current treatment guidelines, which state that the maintenance of remission during pregnancy is essential **(**Carter, et al 2004). Pregnant women should be treated as

surgery, quiescent disease, and CD compared with UC (Bush et al, 2004).

gestational-age infants did not document maternal smoking, alcohol consumption, disease status and medication use.

## **6. Effect of IBD on fertility, pregnancy and foetal outcomes**

## **6.1 IBD and fertility**

Infertility rates in women with inactive CD appear to be similar to those of the general population (8% to 10%) (Khosla et al 1984).

Active disease decreases fertility, possibly as a result of decrease libido, fatigue and concerns regarding the disease. Other factors may be caused by inflammation or adhesions in the fallopian tubes or ovaries, and may cause painful intercourse (Steinlauf & Present DH **2004)**. Some women choose not to bear children secondary to fear of pregnancy (Marri, 2007) and the risk of inheritance of the disease and the possible harmful effect of drug therapy. Patient education preconception may alleviate this fear and may lead to successful pregnancy.

Women who have undergone ileal pouch-anal anastomosis are less fertile. In a recent metaanalysis of ileal pouch-anal anastomosis (IPPPA) resulted in a threefold increased risk of infertility as opposed to those treated medically (Waljee J et al 2006).

Some of the evidence suggests that extensive pelvic surgery and rectal resection with the creation of J pouch lead to scarring, adhesions and tubal infertility (Arkuran & McComb, 2000; Oresland et al, 1994).

Despite these suggestions it appears that the majority of these patients can still conceive with IVF (Kwan & Mahadevan, 2010). In patients with active Crohn's disease there appear to be decreased fertility, which can be restored upon induction of remission (Baiocco & Korelitz, 1984).

It is recommended that female patients who are planning to conceive and require colectomy for acute ulcerative not undergo IPPPA and instead undergo ileostomy and rectal sparing. IPPPA should then be performed after conception. Despite this recommendation, there is no data as yet to support such an approach **(**Janneke, 2010). Patients, who undergo IPPPA, are more likely to have a Caesarean section following RPC (Cornish, 2011).

## **6.2 IBD and pregnancy outcomes**

Due to the lack of prospective studies clinical outcomes in IBD and pregnancy has been controversial with data showing poor outcome and others showing no effect. Some of these reviews or meta-analysis did not take into account some factors such as drug use by the patients, population age, smoking and alcohol and duration of disease (Dominitz et al, 2002; Fonager et al 1992). Similarly the reviews are variable between ulcerative colitis and Crohn's disease.

In 2007 Cornish et al published a meta-analysis on the influence of IBD on pregnancy. The analysis reviewed 12 studies that met specific inclusion criteria and reported the outcomes of interest. The studies included 1952 women with CD, 1113 with UC and 320,531 controls. For women with IBD, the analysis found a 1.87-fold increase in premature births (less than 37 weeks; P<0.001); more than double the incidence of low birth weight (LBW) (less than 2500 g; P<0.001); a 1.5-fold increase in caesarean section (P<0.001); and a 2.37-fold increase in congenital abnormalities (P<0.001). The analysis was unable to determine which women had a higher risk of adverse outcomes, but the authors concluded that pregnant women with IBD should be treated as a high-risk group. There were significant limits to this study. These studies were of an observational nature which make them vulnerable to bias; the low

gestational-age infants did not document maternal smoking, alcohol consumption, disease

Infertility rates in women with inactive CD appear to be similar to those of the general

Active disease decreases fertility, possibly as a result of decrease libido, fatigue and concerns regarding the disease. Other factors may be caused by inflammation or adhesions in the fallopian tubes or ovaries, and may cause painful intercourse (Steinlauf & Present DH **2004)**. Some women choose not to bear children secondary to fear of pregnancy (Marri, 2007) and the risk of inheritance of the disease and the possible harmful effect of drug therapy. Patient education preconception may alleviate this fear and may lead to successful pregnancy. Women who have undergone ileal pouch-anal anastomosis are less fertile. In a recent metaanalysis of ileal pouch-anal anastomosis (IPPPA) resulted in a threefold increased risk of

Some of the evidence suggests that extensive pelvic surgery and rectal resection with the creation of J pouch lead to scarring, adhesions and tubal infertility (Arkuran & McComb,

Despite these suggestions it appears that the majority of these patients can still conceive with IVF (Kwan & Mahadevan, 2010). In patients with active Crohn's disease there appear to be decreased fertility, which can be restored upon induction of remission (Baiocco &

It is recommended that female patients who are planning to conceive and require colectomy for acute ulcerative not undergo IPPPA and instead undergo ileostomy and rectal sparing. IPPPA should then be performed after conception. Despite this recommendation, there is no data as yet to support such an approach **(**Janneke, 2010). Patients, who undergo IPPPA, are

Due to the lack of prospective studies clinical outcomes in IBD and pregnancy has been controversial with data showing poor outcome and others showing no effect. Some of these reviews or meta-analysis did not take into account some factors such as drug use by the patients, population age, smoking and alcohol and duration of disease (Dominitz et al, 2002; Fonager et al 1992). Similarly the reviews are variable between ulcerative colitis and

In 2007 Cornish et al published a meta-analysis on the influence of IBD on pregnancy. The analysis reviewed 12 studies that met specific inclusion criteria and reported the outcomes of interest. The studies included 1952 women with CD, 1113 with UC and 320,531 controls. For women with IBD, the analysis found a 1.87-fold increase in premature births (less than 37 weeks; P<0.001); more than double the incidence of low birth weight (LBW) (less than 2500 g; P<0.001); a 1.5-fold increase in caesarean section (P<0.001); and a 2.37-fold increase in congenital abnormalities (P<0.001). The analysis was unable to determine which women had a higher risk of adverse outcomes, but the authors concluded that pregnant women with IBD should be treated as a high-risk group. There were significant limits to this study. These studies were of an observational nature which make them vulnerable to bias; the low

**6. Effect of IBD on fertility, pregnancy and foetal outcomes** 

infertility as opposed to those treated medically (Waljee J et al 2006).

more likely to have a Caesarean section following RPC (Cornish, 2011).

status and medication use.

2000; Oresland et al, 1994).

**6.2 IBD and pregnancy outcomes** 

Korelitz, 1984).

Crohn's disease.

population (8% to 10%) (Khosla et al 1984).

**6.1 IBD and fertility** 

incidence of adverse outcomes makes statistical precision difficult; and disease activity was not reported in relation to outcomes. In addition, increased risks of congenital abnormalities associated with 5-ASA, azathioprine and anti-TNF-alpha medications seen in a pooled analysis may be associated with the disease and not the medications. The authors also pointed out the need for a definitive study to determine optimal management and to support the development of new guidelines to assist patients and clinicians in decisionmaking **(**Cornish et al 2007).

In a recent publication **by** Raatikainen there was a lower birth weight of newborns from mothers with UC as compared to the general population ( 3317 \_ s.d. 658 g vs. 3506 \_ s.d. 613 g, P = 0.003;3340 \_ s.d. 631 g vs. 3507 \_ s.d. 613 g, P = 0.002) respectively. Newborns in the CD group were also smaller than those in the reference group but the difference was not statistically significant (Raatikainen et al 2011).

A Taiwanese, Asian population database from 2001-2003 study found an increase of preterm births (11.73 vs. 6.25%; p = 0.004) and LBW (12.76 vs. 5.55%; p < 0.001), controlling for maternal characteristics including age, parity and education level (Lin, 2010). In a cohort study from Northern California Kaiser population, pregnant women with IBD were more likely to have a spontaneous abortion (OR: 1.65; 95% CI: 1.09–2.48); an adverse pregnancy outcome (stillbirth, preterm birth or SGA infant; OR: 1.54; 95% CI: 1.00–2.38); or a complication of labour (OR: 1.78; 95% CI: 1.13–2.81). The study did not find a difference in the rate of congenital malformations in control versus IBD patients, either as a group or for UC and CD separately (Mahadevan et al 2007).

In a review in 1998 Subahani et al found that CD, especially active disease, is associated with decreased birth weights, preterm delivery and caesarean section **(**Subahani & Hamilton, 1998)*.* In a case-control study published by Bush comparing 116 pregnancies in patients with IBD with 56,398 controls, there were no differences in ante partum complications, including chronic hypertension, hyperemesis gravidarum, preterm labour or pre-eclampsia. Among patients with IBD, however, induction of labour (32% versus 24%; P=0.002), chorioamnionitis (7% versus 3%; P=0.04), and caesarean section (32% versus 22%; P=0.007) were all more frequent. Neonatal complications, including low birth weight (LBW), very low birth weight, intrauterine growth restriction, Apgar scores and congenital anomalies, were similar in both groups. Subgroup analysis found a decreased risk of LBW associated with previous IBD surgery, quiescent disease, and CD compared with UC (Bush et al, 2004).

The most recent and only prospective study has just been published by the ECCO-Epicom study (Bertoli et al, 2011)**.** This study from Europe was prospective case-controlled with 332 patients with IBD both Crohn's disease and Ulcerative colitis who were pregnant and were compared to non-IBD pregnant women in the general population. In this study there was no difference in live births, spontaneous or therapeutic abortions, infant death in utero, preterm deliveries and caesarean sections. There was no difference in congenital anomalies or birth weight as compared to outcome in pregnancy in the general population. The only risk that was demonstrated to be associated with congenital anomaly and preterm delivery was older age ≥ 35 years. Smoking was found to be a factor which increased the risk of preterm delivery.

Dejaco performed a prospective study assessing risk factors for poor pregnancy outcome in 58 patients with IBD (Dejaco, et al 2006). The authors found that active disease during pregnancy represents a significant risk factor for unfavourable birth outcome. These results support the current treatment guidelines, which state that the maintenance of remission during pregnancy is essential **(**Carter, et al 2004). Pregnant women should be treated as aggressively as women who are not pregnant.

Approach to the Management of the Pregnant

of foetal harm appears remote

evidence of risk in later trimesters)

inflammatory mediators such as TNFα.

taking the drug on a long term basis.

used or are ineffective)

benefit justifies the potential risk to the foetus

**10. Medication and therapy during pregnancy** 

during pregnancy. The FDA categories are listed in **table 2**.

(Osei et al, 1999).

Inflammatory Bowel Disease Patient: Successful Outcome 183

qualified medical personnel to provide a more detailed approximation of risks to the foetus

Aside from Methotrexate and Thalidomide most of the drugs used in IBD appear to be safe during gestation. The US FDA classification of drugs offers a guide to the use of medications

Definition Class

A

B

C

D

X

Controlled studies in women fail to demonstrate a risk to the foetus in the first trimester (and there is no evidence of risk in later trimesters) and the possibility

Either animal reproduction studies have not demonstrated a foetal risk, but there are no controlled studies in pregnant women OR animal reproduction studies have shown an adverse effect (other than decrease in fertility) that was not confirmed in controlled studies in women in the first trimester (and there is no

Either studies in animals have repeated adverse effects on the foetus (teratogenic, embryonic or other) and there are no controlled studies in women or studies in women and animals are not available. Drugs should be given only if the potential

There is positive evidence of human foetal risk but the benefits from use in pregnant women may be acceptable despite the risk (e.g. if the drug is needed in a life threatening situation or for a serious disease for which safer drugs cannot be

Studies in animals or human beings have demonstrated foetal abnormalities OR there is evidence of foetal risk based on human experience OR both, and the risk of the use of the drug in pregnant women clearly outweighs any possible benefit.

Crohn's disease and ulcerative colitis appear to be mediated by different aspects of immune system. CD is thought to be related to the over expression of T cell helper (Th) 1 cytokines, such as TNF-alpha, which stimulate cell-mediated immunity and result in transmural inflammation of the gut (Neissner & Volk, 1995). In contrast, UC is believed to result from a dysregulation of intestinal immunity involving the Th2 cytokine response. Increased expression of TNF-alpha, however, has been observed in patients with UC. The newer treatment modalities, with biologics, over the past decade, are to target specific

There are several medications used to treat IBD. These include aminosalicylates, such as Mesalamine, and immune modulators, such as Azathioprine and Methotrexate. Other drugs include Corticosteroids which are effective in inducing remission in both CD and UC but ineffective in maintenance therapy. Steroids result in side effects in the majority of patients

The drug is contraindicated in women who are or may become pregnant.

Table 2. Food and drug administration (FDA) classes in pregnancy

## **7. Mode of delivery**

The decision to have a caesarean section should be made on purely obstetric grounds with a discussion with the gastroenterologist. In a meta-analysis in 2007, a pregnant woman with IBD was 1.5 times more likely to undergo c section (95% CI 1.26-1.79; p<0.001) (Cornish et al, 2007). Some surgeons advise elective caesarean section to avoid risk of anal sphincter damage. Vaginal delivery and episiotomy may lead to development or worsening of perianal CD (Brandt LJ, 1995). Current indications for caesarean section are active perianal disease and presence of an ileoanal pouch. There is no absolute contraindication to vaginal delivery in pregnant patients with inactive IBD (Alexandra Ilnyckyet al, 1999).

## **8. Endoscopy during pregnancy**

It is best to avoid any major investigation during pregnancy such as radiation, endoscopy or major surgery. Indications for endoscopy during pregnancy include significant or continued gastrointestinal bleeding, dysphagia, severe or (Qureshi et al, 2005) refractory nausea and vomiting or abdominal pain, and a strong suspicion of a colonic mass. It is best to postpone endoscopy to the second trimester. If endoscopy is indicated it is important to place the patient in lateral decubitus position avoid vena caval or aortic compression by the gravid uterus. This otherwise may lead to decreased uterine blood flow and foetal hypoxemia **(**Kemmerer, 1979).

No evidence exists that would suggest endoscopy could affect the pregnancy. Obstetrical support should be available in the event of a pregnancy-related complication, and the presence of foetal heart sounds should be confirmed before administering sedation before and after the procedure (Qureshi et al, 2005).

Endoscopy should be done with minimal sedation. Meperidine, FDA class B followed by small doses of midazolam FDA class C. (Qureshi, 2005) Benzodiazepines (D) should be avoided in the first trimester since they have been associated with congenital cleft palate and when used late in pregnancy with neurobehavioral disorders (Ornoy, 1998; Dolovich, et al 1998; Laegreid et al,1989). Propofol, FDA class B, should be administered by an anaesthetist; its safety as yet in first trimester has not been studied (Gin, 1994).

## **9. Radiology**

An excellent guideline regarding pregnancy and radiation has been published by the CDC Radiation and Pregnancy: A Fact Sheet for Clinicians Prenatal Radiation Exposure: A Fact Sheet for Physicians (CDC) 2003 http://www.bt.cdc.gov/radiation/prenatalphysician.asp , and documentations could be found in: Valentin J, 2000.

In certain circumstances radiographic imaging may be needed to rule out obstruction, perforation or toxic mega colon. Investigations that expose the patient to less radiation are preferable specifically plain abdominal films rather than CT or barium studies. Ultrasound is the safest form of radiologic imaging. It can be used to assess abscess formation and can provide information on bowel wall thickness. MRI studies are also safe and have been used to diagnose terminal ileal CD during pregnancy since active disease in the mother has an adverse effect on the foetus; investigation for diagnostic and therapeutic purposes is warranted and should not be delayed. The foetal radiation dose should be estimated by

The decision to have a caesarean section should be made on purely obstetric grounds with a discussion with the gastroenterologist. In a meta-analysis in 2007, a pregnant woman with IBD was 1.5 times more likely to undergo c section (95% CI 1.26-1.79; p<0.001) (Cornish et al, 2007). Some surgeons advise elective caesarean section to avoid risk of anal sphincter damage. Vaginal delivery and episiotomy may lead to development or worsening of perianal CD (Brandt LJ, 1995). Current indications for caesarean section are active perianal disease and presence of an ileoanal pouch. There is no absolute contraindication to vaginal delivery in pregnant patients with inactive IBD (Alexandra

It is best to avoid any major investigation during pregnancy such as radiation, endoscopy or major surgery. Indications for endoscopy during pregnancy include significant or continued gastrointestinal bleeding, dysphagia, severe or (Qureshi et al, 2005) refractory nausea and vomiting or abdominal pain, and a strong suspicion of a colonic mass. It is best to postpone endoscopy to the second trimester. If endoscopy is indicated it is important to place the patient in lateral decubitus position avoid vena caval or aortic compression by the gravid uterus. This otherwise may lead to decreased uterine blood flow and foetal hypoxemia

No evidence exists that would suggest endoscopy could affect the pregnancy. Obstetrical support should be available in the event of a pregnancy-related complication, and the presence of foetal heart sounds should be confirmed before administering sedation before

Endoscopy should be done with minimal sedation. Meperidine, FDA class B followed by small doses of midazolam FDA class C. (Qureshi, 2005) Benzodiazepines (D) should be avoided in the first trimester since they have been associated with congenital cleft palate and when used late in pregnancy with neurobehavioral disorders (Ornoy, 1998; Dolovich, et al 1998; Laegreid et al,1989). Propofol, FDA class B, should be administered by an

An excellent guideline regarding pregnancy and radiation has been published by the CDC Radiation and Pregnancy: A Fact Sheet for Clinicians Prenatal Radiation Exposure: A Fact Sheet for Physicians (CDC) 2003 http://www.bt.cdc.gov/radiation/prenatalphysician.asp ,

In certain circumstances radiographic imaging may be needed to rule out obstruction, perforation or toxic mega colon. Investigations that expose the patient to less radiation are preferable specifically plain abdominal films rather than CT or barium studies. Ultrasound is the safest form of radiologic imaging. It can be used to assess abscess formation and can provide information on bowel wall thickness. MRI studies are also safe and have been used to diagnose terminal ileal CD during pregnancy since active disease in the mother has an adverse effect on the foetus; investigation for diagnostic and therapeutic purposes is warranted and should not be delayed. The foetal radiation dose should be estimated by

anaesthetist; its safety as yet in first trimester has not been studied (Gin, 1994).

**7. Mode of delivery** 

Ilnyckyet al, 1999).

**(**Kemmerer, 1979).

**9. Radiology** 

**8. Endoscopy during pregnancy** 

and after the procedure (Qureshi et al, 2005).

and documentations could be found in: Valentin J, 2000.

qualified medical personnel to provide a more detailed approximation of risks to the foetus (Osei et al, 1999).

## **10. Medication and therapy during pregnancy**

Aside from Methotrexate and Thalidomide most of the drugs used in IBD appear to be safe during gestation. The US FDA classification of drugs offers a guide to the use of medications during pregnancy. The FDA categories are listed in **table 2**.


Table 2. Food and drug administration (FDA) classes in pregnancy

Crohn's disease and ulcerative colitis appear to be mediated by different aspects of immune system. CD is thought to be related to the over expression of T cell helper (Th) 1 cytokines, such as TNF-alpha, which stimulate cell-mediated immunity and result in transmural inflammation of the gut (Neissner & Volk, 1995). In contrast, UC is believed to result from a dysregulation of intestinal immunity involving the Th2 cytokine response. Increased expression of TNF-alpha, however, has been observed in patients with UC. The newer treatment modalities, with biologics, over the past decade, are to target specific inflammatory mediators such as TNFα.

There are several medications used to treat IBD. These include aminosalicylates, such as Mesalamine, and immune modulators, such as Azathioprine and Methotrexate. Other drugs include Corticosteroids which are effective in inducing remission in both CD and UC but ineffective in maintenance therapy. Steroids result in side effects in the majority of patients taking the drug on a long term basis.

Approach to the Management of the Pregnant

effect is reversed upon discontinuing the drug.

(Mahadevan &Corley, 2006).

Norgard et al, 2003).

Inflammatory Bowel Disease Patient: Successful Outcome 185

outcome was similar to that expected in the general population (Jarnerot, 1982). Sulfapyridine acts as a competitive inhibitor of the enzyme dihydropteroate synthase in the folate metabolism; and this may lead to a deficiency of dihydrofolate and tetrahydrofolate.

Oligospermia and infertility have been observed in men treated with Sulfasalazine; this

At the 2006 Digestive Disease Week in Los Angeles, USA, Mahadevan reported that the use of 5-ASA and Sulfasalazine during pregnancy was not associated with an increase in adverse outcomes. A trend toward an increased risk of congenital malformations was seen during conception and the first trimester with Sulfasalazine, but not with 5-ASA. On the contrary, an increased risk of adverse outcomes was seen in women not taking 5-ASA during the second and third trimesters, suggesting a protective effect of the medication

In a recent meta-analysis of seven studies prior to 2007, with a total of 2200 pregnant women with IBD there was an 1.16-fold increase in congenital malformations, an 2.38-fold increase in stillbirth, an 1.14-fold increase in spontaneous abortion, an 1.35-fold increase in preterm delivery, and an 0.93-fold increase in low birth weight (Rahimi R et al 2008;

Asacol is covered with an inactive enteric coating of dibutyl phthalate (DBP) that prevents the medication from degrading before it reaches the small intestine. DBP was associated with external and skeletal malformations and adverse effects on the male reproductive of rodents system. Patients who are using Asacol have 50 times higher mean urinary concentration of monobutyl phthalate, the main DBP metabolite, than the mean for nonusers (2,257 microg/L vs. 46 microg/L; p < 0.0001) (Hernández-Díaz et al 2009). These results raise concern about potential human health risks, particularly pregnant women and children. Although this has not been shown in any human study, Asacol should be used during pregnancy only if the potential benefit justifies the potential risk to the fetus.FDA 2010 *Safety Labelling Changes Approved By FDA Center for Drug Evaluation and Research (CDER) – May 2010.These patients may need to be switched to non DBP containing Mesalamine .*

Contraindicated Limited data, potential toxicity Safe

Tacolimus (FK506) Azathioprine Acid

Table 4. Breastfeeding safety of medications used to treat IBD

 Adalimumab Infliximab

**12.1 Azathioprine/6 mercaptopurine** 

**12. Immune modulators** 

Thalidomide Metronidazole 5-ASA preparations

Methotrexate Fluoroquinolones (sulfasalazine/mesalamine) Cyclosporine Bisphosphonates Amoxacillin/clavulanic

6-mercaptopurine (6MP) and its prodrug azathioprine (AZA) are pregnancy category D drugs. These have been proven to be effective in the treatment of steroid-dependent or resistant IBD Crohn's disease (CD) and ulcerative colitis (Gisbert et al, 2009). These drugs

6-Mercaptopurine (6-MP) Corticosteroids

Patients receiving Sulfasalazine should receive folic acid supplementations.


Table 3. Inflammatory bowel disease medications FDA classes: Summary of safety data during pregnancy\* *Asacol (Mesalamine)* Dibutyl phthalate (DBP) **P=PRECAUTIONS**

A recent study by Moskovitz et al (2004) assessed the effect of 5-ASA drugs, metronidazole, ciprofloxacin, prednisone, 6-mercaptopurine, azathioprine and cyclosporine on pregnancy outcomes in 113 IBD patients with 207 documented conceptions. The authors reviewed information obtained on smoking history, birth weight, and type of delivery. They also followed the pregnancy outcomes: spontaneous abortion, therapeutic abortion, maternal or foetal illness resulting in abortion, premature birth, healthy full-term birth, multiple births, ectopic pregnancy and congenital defects. The study also analyzed the effect of medications on pregnancy outcomes during the first trimester and at any time during the pregnancy. No significant differences were seen among groups in pregnancy outcomes. A multivariate analysis controlling for maternal age showed no negative influence of any medication on pregnancy outcomes.

## **11. 5-ASA compounds sulfasalazine/mesalamine/olsalazine**

Sulfasalazine has been assigned to pregnancy category B by the FDA. It was one of the earliest therapeutics used in IBD. Sulfasalazine is metabolized by intestinal bacterial flora to sulfapyridine (SP) and 5-ASA. Sulfapyridine crosses the placenta to the fetus with the foetal concentrations being approximately the same as the maternal serum concentrations. Caution should be exercised when Sulfasalazine is administered to a nursing woman. Sulphonamides are excreted in the milk. In the newborn, they compete with bilirubin for binding sites on the plasma proteins and may thus cause kernicterus.

There was no significant increase in prevalence of selected congenital abnormalities in the children of women treated with Sulfasalazine during pregnancy. A review of the medical literature covering 1,155 pregnancies in women with ulcerative colitis suggested that the

**Medications** FDA

Azathioprine; 6-MP <sup>D</sup>

Methotrexate; thalidomide <sup>X</sup>

A recent study by Moskovitz et al (2004) assessed the effect of 5-ASA drugs, metronidazole, ciprofloxacin, prednisone, 6-mercaptopurine, azathioprine and cyclosporine on pregnancy outcomes in 113 IBD patients with 207 documented conceptions. The authors reviewed information obtained on smoking history, birth weight, and type of delivery. They also followed the pregnancy outcomes: spontaneous abortion, therapeutic abortion, maternal or foetal illness resulting in abortion, premature birth, healthy full-term birth, multiple births, ectopic pregnancy and congenital defects. The study also analyzed the effect of medications on pregnancy outcomes during the first trimester and at any time during the pregnancy. No significant differences were seen among groups in pregnancy outcomes. A multivariate analysis controlling for maternal age showed no negative influence of any medication on

Sulfasalazine has been assigned to pregnancy category B by the FDA. It was one of the earliest therapeutics used in IBD. Sulfasalazine is metabolized by intestinal bacterial flora to sulfapyridine (SP) and 5-ASA. Sulfapyridine crosses the placenta to the fetus with the foetal concentrations being approximately the same as the maternal serum concentrations. Caution should be exercised when Sulfasalazine is administered to a nursing woman. Sulphonamides are excreted in the milk. In the newborn, they compete with bilirubin for

There was no significant increase in prevalence of selected congenital abnormalities in the children of women treated with Sulfasalazine during pregnancy. A review of the medical literature covering 1,155 pregnancies in women with ulcerative colitis suggested that the

Table 3. Inflammatory bowel disease medications FDA classes: Summary of safety

5-Aminosalicylic acid\* preparations (sulfasalazine,

5-Aminosalicylic acid preparations (Olsalazine);

data during pregnancy\* *Asacol (Mesalamine)* Dibutyl phthalate (DBP)

**11. 5-ASA compounds sulfasalazine/mesalamine/olsalazine** 

binding sites on the plasma proteins and may thus cause kernicterus.

amoxicillin/clavulanic acid; infliximab; adalimumab;

mesalamine, balsalazide); metronidazole,

fluoroquinolones; corticosteroids; bisphosphonates;cyclosporin; tacrolimus

Certolizumab

Biphosphonates

**P=PRECAUTIONS**

pregnancy outcomes.

Class

B

C

outcome was similar to that expected in the general population (Jarnerot, 1982). Sulfapyridine acts as a competitive inhibitor of the enzyme dihydropteroate synthase in the folate metabolism; and this may lead to a deficiency of dihydrofolate and tetrahydrofolate. Patients receiving Sulfasalazine should receive folic acid supplementations.

Oligospermia and infertility have been observed in men treated with Sulfasalazine; this effect is reversed upon discontinuing the drug.

At the 2006 Digestive Disease Week in Los Angeles, USA, Mahadevan reported that the use of 5-ASA and Sulfasalazine during pregnancy was not associated with an increase in adverse outcomes. A trend toward an increased risk of congenital malformations was seen during conception and the first trimester with Sulfasalazine, but not with 5-ASA. On the contrary, an increased risk of adverse outcomes was seen in women not taking 5-ASA during the second and third trimesters, suggesting a protective effect of the medication (Mahadevan &Corley, 2006).

In a recent meta-analysis of seven studies prior to 2007, with a total of 2200 pregnant women with IBD there was an 1.16-fold increase in congenital malformations, an 2.38-fold increase in stillbirth, an 1.14-fold increase in spontaneous abortion, an 1.35-fold increase in preterm delivery, and an 0.93-fold increase in low birth weight (Rahimi R et al 2008; Norgard et al, 2003).

Asacol is covered with an inactive enteric coating of dibutyl phthalate (DBP) that prevents the medication from degrading before it reaches the small intestine. DBP was associated with external and skeletal malformations and adverse effects on the male reproductive of rodents system. Patients who are using Asacol have 50 times higher mean urinary concentration of monobutyl phthalate, the main DBP metabolite, than the mean for nonusers (2,257 microg/L vs. 46 microg/L; p < 0.0001) (Hernández-Díaz et al 2009). These results raise concern about potential human health risks, particularly pregnant women and children. Although this has not been shown in any human study, Asacol should be used during pregnancy only if the potential benefit justifies the potential risk to the fetus.FDA 2010 *Safety Labelling Changes Approved By FDA Center for Drug Evaluation and Research (CDER) – May 2010.These patients may need to be switched to non DBP containing Mesalamine .*


Table 4. Breastfeeding safety of medications used to treat IBD

## **12. Immune modulators**

#### **12.1 Azathioprine/6 mercaptopurine**

6-mercaptopurine (6MP) and its prodrug azathioprine (AZA) are pregnancy category D drugs. These have been proven to be effective in the treatment of steroid-dependent or resistant IBD Crohn's disease (CD) and ulcerative colitis (Gisbert et al, 2009). These drugs

Approach to the Management of the Pregnant

(Van Assche, et al 2010).

Doctor and partner.

Inflammatory Bowel Disease Patient: Successful Outcome 187

In a prospective, controlled, multicentre study conducted by the Tel Aviv University (Israel), 189 pregnant women on azathioprine were compared to a cohort of 230 pregnant women who did not take Azathioprine*.* The aim of that study was to determine risk of congenital malformation in pregnant women exposed to azathioprine and to assess the pregnancy outcome (Goldstein, et al, 2007). The rate of congenital malformations did not differ between the two groups; there were more cases noted of prematurity (21% vs. 5%, *P* <

The most recent ECCO consensus guidelines recommend the use of AZA/6-MP in high risk patients where the benefit outweighs the risk of relapse of the disease. The European consensus guidelines consider azathioprine to be safe and well tolerated in pregnant women with no consistent reports of abnormalities of fertility, prematurity, or congenital defects

Based on the data published in the transplant literature, the American Gastroenterology Association, and The ECCO consensus guidelines, the recommendation is that Azathioprine /6-Mercaptopurine treatment to be continued during pregnancy (Van Assche, et al 2010). The discontinuation of therapy in pregnant women, in remission, may be more harmful (Zlatanic et al, 2003) than precipitating a flare up with more deleterious effect on the neonate ( Mahadevan U, Kane S. 2006). Although some negative outcomes in some cases of pregnancy in IBD women have been reported, the majority of case series or cohort studies have not shown an increase in congenital anomalies. Despite this recommendation, the risks and benefits of treatment must be carefully balanced by the patient in consultation with her

The majority of physicians have not recommended breast feeding of neonates while mothers are receiving thiopurines. The reason for this has been the theoretical potential risks of bone marrow suppression, susceptibility to infection, and pancreatitis in the neonate. In a prospective study in 10 women receiving AZA while breast feeding in 31 samples, there was only one woman who had a low measurable level on AZA in two samples of the breast milk. The concentrations of 6-MP was 1.2 and 7.6 nanograms/mL, as compared with therapeutic immunosuppressant level of 50 nanograms/mL in serum. The conclusion of the authors was that there were no clinical or haematological signs of immunosuppression in any of the ten neonates and that breast feeding should not be withheld (Sau et al, 2007). Based on the case reports and the presence of low to absent levels of Thiopurine in the breast milk of women receiving thiopurines, breast feeding can be continued. The benefits of breastfeeding outweigh the theoretical risk. A discussion with the mother explaining the

Pregnant women with IBD who are on thiopurines drugs, and who are in remission should continue their medication during their pregnancy (Caprilli, et al 2006; Gisbert et al 2009; Van Assche et al, 2010). The safety of Thiopurine derivatives has been controversial in male patients exposed to thiopurines at the time of conception. In a retrospective study by Rajapakse published in 2000, there was an increase in adverse side effects. In 13 males

0.001) and low birth weight (23% vs. 6%, *P* < 0.001) in the azathioprine group.

**14. Breast feeding: Azathioprine/6 mercaptopurine** 

risk benefit should be undertaken (*Gardiner al, 2006; Christensen, et al 2008*)*.* 

**15. Birth outcome in IBD fathers on immunosuppressive drugs** 

are used as immunosuppressive therapies in autoimmune diseases, transplant patients and in leukemia. Azathioprine and 6-mercaptopurine are purine analogues that interfere with the synthesis of adenine and guanine ribonucleosides. These ribonucleosides are important precursors of DNA and RNA and hence act on rapidly producing cells. Following oral intake of AZA 47% of the drug is available to the systemic circulation whereas only 16% of 6-mercaptopurine is available (Zimm, et al, 1983). In animal studies, in mice after intraperitoneal administration of azathioprine, there was evidence of increased frequencies of cleft palate, open-eye, and skeletal anomalies. There was a significant decrease in thymic size. The dose given is equivalent of 4–13 times the maximum human therapeutic dose of AZA.There was no discussion regarding its effect on the immune system development, which is important for future immunity.

The safety of azathioprine in pregnancy comes from studies in transplantation and rheumatology patients (Rosenkrantz et al, 1967.; Gaudier, et al 1988).

The foetus is protected from potential teratogenic effects of azathioprine and 6-MP due to the lack in foetal liver of the enzyme inosinate phosphorylase which is necessary to convert azathioprine and 6-MP to active metabolites. Both these medications when used in small doses in clinical practice do not affect human interstitial cell function or gametogenesis (Golby, 1970; Penn, et al 1971).

#### **13. Should azathioprine/6 mercaptopurine be used in pregnancy?**

In a retrospective chart review and telephone recalls of some of the patients who had received 6-Mercaptopurine for IBD before or during conception, there was no statistical difference in abortion secondary to a birth defect, major congenital malformations, neoplasia, or increased infections among male or female patients taking 6-MP compared with controls (RR = 0.85 [0.47-1.55], P = 0.59) (Francella, et al, 2003).

In a retrospective Swedish registry study reviewing patients receiving azathioprine (AZA) during pregnancy by women, the majority of whom were with inflammatory bowel disease, and the rest being with other autoimmune disorders, malignancy, and organ transplantation, the rate of congenital malformations was 6.2% in the AZA group and 4.7% among all infants born (adjusted OR: 1.42, 95% CI: 0.98–2.04) (Cleary& Kallen, 2009).

There was an association between early pregnancy AZA exposure and ventricular/atrial septal defects (adjusted OR: 3.18, 95% CI: 1.45–6.04). Exposed infants were also more likely to be preterm, to weigh <2500 gm, and to be small for gestational age compared to all infants born. The authors pointed in their article regarding the severity of the disease and its association with drug use (Cleary& Kallen, 2009).

 In a Danish registry study in women with Crohn's diseases receiving drug therapy during pregnancy the risk of preterm birth and congenital abnormalities was greater when azathioprine/mercaptopurine was prescribed as compared to women who did not used drugs. Preterm births were more prevalent among Thiopurine-exposed women (25%) compared to the reference group (6.5%). Congenital abnormalities were also more prevalent among azathioprine/mercaptopurine-exposed women (15.4 versus 5.7%, adjusted relative risk, 2.9; 95% CI, 0.9–8.9). Among Thiopurine-exposed women, the risk of preterm birth was also increased to 4.2 (95% CI, 1.4–12.5) compared to the control group (Norgard, et al 2007). The conclusion of this study was subsequently challenged, based on bias, the disease activity of the patients and the use of confidential interval **(**Simpson, et al 2008).

are used as immunosuppressive therapies in autoimmune diseases, transplant patients and in leukemia. Azathioprine and 6-mercaptopurine are purine analogues that interfere with the synthesis of adenine and guanine ribonucleosides. These ribonucleosides are important precursors of DNA and RNA and hence act on rapidly producing cells. Following oral intake of AZA 47% of the drug is available to the systemic circulation whereas only 16% of 6-mercaptopurine is available (Zimm, et al, 1983). In animal studies, in mice after intraperitoneal administration of azathioprine, there was evidence of increased frequencies of cleft palate, open-eye, and skeletal anomalies. There was a significant decrease in thymic size. The dose given is equivalent of 4–13 times the maximum human therapeutic dose of AZA.There was no discussion regarding its effect on the immune system development,

The safety of azathioprine in pregnancy comes from studies in transplantation and

The foetus is protected from potential teratogenic effects of azathioprine and 6-MP due to the lack in foetal liver of the enzyme inosinate phosphorylase which is necessary to convert azathioprine and 6-MP to active metabolites. Both these medications when used in small doses in clinical practice do not affect human interstitial cell function or gametogenesis

In a retrospective chart review and telephone recalls of some of the patients who had received 6-Mercaptopurine for IBD before or during conception, there was no statistical difference in abortion secondary to a birth defect, major congenital malformations, neoplasia, or increased infections among male or female patients taking 6-MP compared

In a retrospective Swedish registry study reviewing patients receiving azathioprine (AZA) during pregnancy by women, the majority of whom were with inflammatory bowel disease, and the rest being with other autoimmune disorders, malignancy, and organ transplantation, the rate of congenital malformations was 6.2% in the AZA group and 4.7%

There was an association between early pregnancy AZA exposure and ventricular/atrial septal defects (adjusted OR: 3.18, 95% CI: 1.45–6.04). Exposed infants were also more likely to be preterm, to weigh <2500 gm, and to be small for gestational age compared to all infants born. The authors pointed in their article regarding the severity of the disease and its

 In a Danish registry study in women with Crohn's diseases receiving drug therapy during pregnancy the risk of preterm birth and congenital abnormalities was greater when azathioprine/mercaptopurine was prescribed as compared to women who did not used drugs. Preterm births were more prevalent among Thiopurine-exposed women (25%) compared to the reference group (6.5%). Congenital abnormalities were also more prevalent among azathioprine/mercaptopurine-exposed women (15.4 versus 5.7%, adjusted relative risk, 2.9; 95% CI, 0.9–8.9). Among Thiopurine-exposed women, the risk of preterm birth was also increased to 4.2 (95% CI, 1.4–12.5) compared to the control group (Norgard, et al 2007). The conclusion of this study was subsequently challenged, based on bias, the disease activity of the patients and the use of confidential interval

among all infants born (adjusted OR: 1.42, 95% CI: 0.98–2.04) (Cleary& Kallen, 2009).

rheumatology patients (Rosenkrantz et al, 1967.; Gaudier, et al 1988).

with controls (RR = 0.85 [0.47-1.55], P = 0.59) (Francella, et al, 2003).

association with drug use (Cleary& Kallen, 2009).

**(**Simpson, et al 2008).

**13. Should azathioprine/6 mercaptopurine be used in pregnancy?** 

which is important for future immunity.

(Golby, 1970; Penn, et al 1971).

In a prospective, controlled, multicentre study conducted by the Tel Aviv University (Israel), 189 pregnant women on azathioprine were compared to a cohort of 230 pregnant women who did not take Azathioprine*.* The aim of that study was to determine risk of congenital malformation in pregnant women exposed to azathioprine and to assess the pregnancy outcome (Goldstein, et al, 2007). The rate of congenital malformations did not differ between the two groups; there were more cases noted of prematurity (21% vs. 5%, *P* < 0.001) and low birth weight (23% vs. 6%, *P* < 0.001) in the azathioprine group.

The most recent ECCO consensus guidelines recommend the use of AZA/6-MP in high risk patients where the benefit outweighs the risk of relapse of the disease. The European consensus guidelines consider azathioprine to be safe and well tolerated in pregnant women with no consistent reports of abnormalities of fertility, prematurity, or congenital defects (Van Assche, et al 2010).

Based on the data published in the transplant literature, the American Gastroenterology Association, and The ECCO consensus guidelines, the recommendation is that Azathioprine /6-Mercaptopurine treatment to be continued during pregnancy (Van Assche, et al 2010). The discontinuation of therapy in pregnant women, in remission, may be more harmful (Zlatanic et al, 2003) than precipitating a flare up with more deleterious effect on the neonate ( Mahadevan U, Kane S. 2006). Although some negative outcomes in some cases of pregnancy in IBD women have been reported, the majority of case series or cohort studies have not shown an increase in congenital anomalies. Despite this recommendation, the risks and benefits of treatment must be carefully balanced by the patient in consultation with her Doctor and partner.

## **14. Breast feeding: Azathioprine/6 mercaptopurine**

The majority of physicians have not recommended breast feeding of neonates while mothers are receiving thiopurines. The reason for this has been the theoretical potential risks of bone marrow suppression, susceptibility to infection, and pancreatitis in the neonate. In a prospective study in 10 women receiving AZA while breast feeding in 31 samples, there was only one woman who had a low measurable level on AZA in two samples of the breast milk. The concentrations of 6-MP was 1.2 and 7.6 nanograms/mL, as compared with therapeutic immunosuppressant level of 50 nanograms/mL in serum. The conclusion of the authors was that there were no clinical or haematological signs of immunosuppression in any of the ten neonates and that breast feeding should not be withheld (Sau et al, 2007).

Based on the case reports and the presence of low to absent levels of Thiopurine in the breast milk of women receiving thiopurines, breast feeding can be continued. The benefits of breastfeeding outweigh the theoretical risk. A discussion with the mother explaining the risk benefit should be undertaken (*Gardiner al, 2006; Christensen, et al 2008*)*.* 

## **15. Birth outcome in IBD fathers on immunosuppressive drugs**

Pregnant women with IBD who are on thiopurines drugs, and who are in remission should continue their medication during their pregnancy (Caprilli, et al 2006; Gisbert et al 2009; Van Assche et al, 2010). The safety of Thiopurine derivatives has been controversial in male patients exposed to thiopurines at the time of conception. In a retrospective study by Rajapakse published in 2000, there was an increase in adverse side effects. In 13 males

Approach to the Management of the Pregnant

precipitated, the benefit outweighs the risk.

to be effective for induction of remission in Crohn's disease.

prednisolone or equivalent (Ferguson et al, 2008; Blanford et al, 1977).

**19. Immunobiologic therapy, pregnancy and lactation** 

controlled studies of pregnant women have not been conducted.

during breast-feeding is currently advocated.

no evidence of foetal abnormality (Beaulieu, et al 2009).

defects (Mogadam et al,1981).

**Breast feeding: Corticosteroids** 

**Budesonides and Breast feeding** 

al, 2007).

**18. Budesonide** 

Inflammatory Bowel Disease Patient: Successful Outcome 189

any more likely to have offspring with a cleft lip with or without cleft palate than mothers who did not take the drug (Anders et al, 2011). A prospective study was published by Mogadam reporting on the use of corticosteroids during pregnancy which was used with or without Sulfasalazine. In that study they followed the outcome in two hundred eighty-seven pregnancies on treatment and compared to 244 pregnant IBD on no treatment. There was no increased incidence of prematurity, spontaneous abortion, stillbirth, or developmental

Pregnant women with active disease during conception can be safely treated with corticosteroids to induce remission. Although gestational diabetes and hypertension may be

Budesonide is an enteric coated locally acting glucocorticoid preparation which has a pHand time-dependent coating that enables its release into the ileum and ascending colon. It is used for the treatment of mild to moderate Crohn's disease with limited systemic bioavailability due to extensive first-pass hepatic metabolism. Budesonide has been shown

In a limited study using 8 patients with Crohn's disease at a dose of 6-9 mg daily there was

Corticosteroids are secreted in small amounts into breast milk. The maternal: foetal ratio of steroid serum concentrations depends on which steroid the patient is taking. The foetal levels are 10%-12% of that in maternal serum. It has been suggested that infants should be monitored for adrenal suppression if the mothers are taking a more than a daily dose of 40 mg. While breastfeeding is safe with steroid use, mothers are encouraged to defer breastfeeding until 4 h after taking oral dosing of steroids to reduce neonatal exposure. However, systemic effects in the infant are unlikely with doses of up to 40mg of

Limited studies are available on budesonide in breast feeding. In a study in asthmatic nursing women on maintenance treatment with inhaled budesonide (200 or 400 microg twice daily) showed negligible systemic exposure to budesonide in breas**t**-fed infants (Fält et

Anti-TNF agents fall within the US FDA category B concerning foetal risk, because animal reproduction studies have failed to demonstrate a risk to the foetus but adequate and well-

Biological therapies targeting TNF-α have significantly improved the management of IBD refractory to conventional therapies, in steroids sparing, improvement in perianal disease and maintaining remission. It has been shown to result in mucosal healing. Data regarding their safety are scarce for the other compounds. No increased risks associated with pregnancy have been observed for Infliximab or Adalumimab, but caution in pregnancy and

exposed to Thiopurine at the time of conception, there were two congenital anomalies and two spontaneous abortions. Based on that study there was some concern about continuing the drug in male patients wishing to have children. Further publications did not confirm these findings (Francella, et al, 2003; Truel, et al 2010).

## **16. Cyclosporine and inflammatory bowel disease**

Cyclosporine (CsA) is classified as FDA pregnancy category C. CsA is a selective immunosuppressive drug that has been used mainly in solid organ transplant such as liver, kidney and heart. CsA inhibits the activation of T cells, preventing formation of IL-2.In inflammatory bowel disease, it is mostly used to induce remission in acute ulcerative colitis non responsive to conventional therapy intravenous corticosteroids (Lichtiger et al, 1994; Cohen et al, 1999). Its use has been mostly used to delay surgery in acute ulcerative colitis. The majority of literature in cyclosporine and pregnancy has been reported in transplant publications (Nagy et al, 2003).

Most of the data regarding the use of cyclosporine in pregnancy comes from transplant patients. In a meta-analysis of 15 studies of pregnancy outcomes in 410 transplant patients receiving cyclosporine, showed no significant increase in major malformations. Congenital malformation was 4.1% which was similar to the general population receiving no drugs. (Nagy et al, 2003). 2003). Pregnant transplant patients who are stable while receiving Cyclosporine appear to have a good pregnancy and foetal outcome Nagy et al, 2003). Cyclosporine is secreted in breast milk at high concentrations. Due to its possible toxic effect on the newborn and possible immunosuppression, the American Paediatric association does not recommended breast feeding while taking the drug (Kwan et al, 2010).

## **17. Corticosteroids (FDA class C)**

Corticosteroids are classified as pregnancy category C drugs. They are given to patients via a variety of forms which include oral and topical formulations and those of parental preparations. These include prednisone, prednisolone, dexamethasone and budesonide. Corticosteroids are frequently used to induce remission in both Crohn's disease and ulcerative colitis. These drugs have not been shown to be effective in maintenance therapy although they are associated with side effects in almost 100% of patients taking these medications for a long term *(*Alstead & Nelson, 2003). The foetal placenta contains the enzyme 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2), which catalyzes the metabolism of cortisol and corticosterone to inert 11-keto forms (cortisone, 11-dehydrocorticosterone). This placental enzymic barrier allows the maternal cortisol to be inactivated so that the majority of cortisol in the human fetal circulation at term is derived from the fetal adrenals (Murphy et al ,1974; Lopez-Bernal et al,1980; Beitens, et al 1973; Stewart et al, 1995). Dexamethasone is not inactivated by 11β-hydroxysteroid dehydrogenase hence it passes the placenta freely and thus should be avoided during conception.

Corticosteroids have been implicated in oral cleft palate when they are used in the first trimester (Bush et al, 2004; Carmichael, et al 1999; Pradat et al, 2003).

A recent nationwide health registry data study from Denmark between 1996 and 2008, looking at pregnant women receiving corticosteroids, with 832,637 live births was recently published in the Canadian Medical Association journal (CMAJ). This study did not demonstrate that pregnant women receiving corticosteroids during the first trimester were any more likely to have offspring with a cleft lip with or without cleft palate than mothers who did not take the drug (Anders et al, 2011). A prospective study was published by Mogadam reporting on the use of corticosteroids during pregnancy which was used with or without Sulfasalazine. In that study they followed the outcome in two hundred eighty-seven pregnancies on treatment and compared to 244 pregnant IBD on no treatment. There was no increased incidence of prematurity, spontaneous abortion, stillbirth, or developmental defects (Mogadam et al,1981).

Pregnant women with active disease during conception can be safely treated with corticosteroids to induce remission. Although gestational diabetes and hypertension may be precipitated, the benefit outweighs the risk.

## **18. Budesonide**

188 Inflammatory Bowel Disease – Advances in Pathogenesis and Management

exposed to Thiopurine at the time of conception, there were two congenital anomalies and two spontaneous abortions. Based on that study there was some concern about continuing the drug in male patients wishing to have children. Further publications did not confirm

Cyclosporine (CsA) is classified as FDA pregnancy category C. CsA is a selective immunosuppressive drug that has been used mainly in solid organ transplant such as liver, kidney and heart. CsA inhibits the activation of T cells, preventing formation of IL-2.In inflammatory bowel disease, it is mostly used to induce remission in acute ulcerative colitis non responsive to conventional therapy intravenous corticosteroids (Lichtiger et al, 1994; Cohen et al, 1999). Its use has been mostly used to delay surgery in acute ulcerative colitis. The majority of literature in cyclosporine and pregnancy has been reported in transplant

Most of the data regarding the use of cyclosporine in pregnancy comes from transplant patients. In a meta-analysis of 15 studies of pregnancy outcomes in 410 transplant patients receiving cyclosporine, showed no significant increase in major malformations. Congenital malformation was 4.1% which was similar to the general population receiving no drugs. (Nagy et al, 2003). 2003). Pregnant transplant patients who are stable while receiving Cyclosporine appear to have a good pregnancy and foetal outcome Nagy et al, 2003). Cyclosporine is secreted in breast milk at high concentrations. Due to its possible toxic effect on the newborn and possible immunosuppression, the American Paediatric association does

Corticosteroids are classified as pregnancy category C drugs. They are given to patients via a variety of forms which include oral and topical formulations and those of parental preparations. These include prednisone, prednisolone, dexamethasone and budesonide. Corticosteroids are frequently used to induce remission in both Crohn's disease and ulcerative colitis. These drugs have not been shown to be effective in maintenance therapy although they are associated with side effects in almost 100% of patients taking these medications for a long term *(*Alstead & Nelson, 2003). The foetal placenta contains the enzyme 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2), which catalyzes the metabolism of cortisol and corticosterone to inert 11-keto forms (cortisone, 11-dehydrocorticosterone). This placental enzymic barrier allows the maternal cortisol to be inactivated so that the majority of cortisol in the human fetal circulation at term is derived from the fetal adrenals (Murphy et al ,1974; Lopez-Bernal et al,1980; Beitens, et al 1973; Stewart et al, 1995). Dexamethasone is not inactivated by 11β-hydroxysteroid dehydrogenase hence it passes the placenta freely and thus

Corticosteroids have been implicated in oral cleft palate when they are used in the first

A recent nationwide health registry data study from Denmark between 1996 and 2008, looking at pregnant women receiving corticosteroids, with 832,637 live births was recently published in the Canadian Medical Association journal (CMAJ). This study did not demonstrate that pregnant women receiving corticosteroids during the first trimester were

trimester (Bush et al, 2004; Carmichael, et al 1999; Pradat et al, 2003).

not recommended breast feeding while taking the drug (Kwan et al, 2010).

these findings (Francella, et al, 2003; Truel, et al 2010).

publications (Nagy et al, 2003).

**17. Corticosteroids (FDA class C)** 

should be avoided during conception.

**16. Cyclosporine and inflammatory bowel disease** 

Budesonide is an enteric coated locally acting glucocorticoid preparation which has a pHand time-dependent coating that enables its release into the ileum and ascending colon. It is used for the treatment of mild to moderate Crohn's disease with limited systemic bioavailability due to extensive first-pass hepatic metabolism. Budesonide has been shown to be effective for induction of remission in Crohn's disease.

In a limited study using 8 patients with Crohn's disease at a dose of 6-9 mg daily there was no evidence of foetal abnormality (Beaulieu, et al 2009).

#### **Breast feeding: Corticosteroids**

Corticosteroids are secreted in small amounts into breast milk. The maternal: foetal ratio of steroid serum concentrations depends on which steroid the patient is taking. The foetal levels are 10%-12% of that in maternal serum. It has been suggested that infants should be monitored for adrenal suppression if the mothers are taking a more than a daily dose of 40 mg. While breastfeeding is safe with steroid use, mothers are encouraged to defer breastfeeding until 4 h after taking oral dosing of steroids to reduce neonatal exposure. However, systemic effects in the infant are unlikely with doses of up to 40mg of prednisolone or equivalent (Ferguson et al, 2008; Blanford et al, 1977).

#### **Budesonides and Breast feeding**

Limited studies are available on budesonide in breast feeding. In a study in asthmatic nursing women on maintenance treatment with inhaled budesonide (200 or 400 microg twice daily) showed negligible systemic exposure to budesonide in breas**t**-fed infants (Fält et al, 2007).

## **19. Immunobiologic therapy, pregnancy and lactation**

Anti-TNF agents fall within the US FDA category B concerning foetal risk, because animal reproduction studies have failed to demonstrate a risk to the foetus but adequate and wellcontrolled studies of pregnant women have not been conducted.

Biological therapies targeting TNF-α have significantly improved the management of IBD refractory to conventional therapies, in steroids sparing, improvement in perianal disease and maintaining remission. It has been shown to result in mucosal healing. Data regarding their safety are scarce for the other compounds. No increased risks associated with pregnancy have been observed for Infliximab or Adalumimab, but caution in pregnancy and during breast-feeding is currently advocated.

Approach to the Management of the Pregnant

**20. Bisphosphonates (FDA Class C)** 

biphosphonates should be carefully weighed in these women.

al, 2011).

et al, 2007).

two trimesters of pregnancy (Djokanovic et al, 2011).

Inflammatory Bowel Disease Patient: Successful Outcome 191

Recently, the mother risk program in Canada published a review of 300 pregnancies in women who received anti TNF alpha during conception. The review suggested that infliximab carries low foetal risk and is compatible with use during conception and the first

A recent observational study by the Leuven, group in Belgium, was published. They assessed pregnancy outcomes in 212 women with IBD. Pregnancy outcome was assessed in 42 pregnancies in women who received anti-TNF treatment (35 IFX, 7 ADA) and were compared with that in 23 pregnancies prior to IBD diagnosis, 78 pregnancies before start of IFX, 53 pregnancies with indirect exposure to IFX, and 56 matched pregnancies in healthy women. Their conclusion was that direct exposure to anti-TNF treatment during pregnancy was not related to a higher incidence of adverse outcomes than IBD overall (Schnitzler et

Vasiliauskas monitored a pregnant patient who continued on standard-dose infliximab therapy during her pregnancy and lactation. The measurement of serum infliximab in the breastfed infant was 39.5 μg/mL or less and the drug was not detected in the breast milk. Importantly, despite continued breast-feeding and adherence to therapy, the levels declined in the infant over the subsequent six months. They concluded that infliximab levels were likely due to placental transfer and less likely the result of breastfeeding (Vasiliauskas et al, 2006). The blood levels in the newborn were within the therapeutic range. The authors recommended that such therapy be avoided after 30 weeks gestation when possible. As of the date of this publication, no increased risk of embryotoxicity, teratogenicity or adverse pregnancy outcome has been reported in patients treated with anti-TNF therapy (Skomsvoll

The majority of studies regarding breast feeding while receiving anti-TNF therapy has not demonstrated any adverse outcomes. The data, however, appear limited. In a case report publication in which serial sampling of breast milk over a one-month time period failed to detect any concentrations of infliximab. Furthermore, at a follow-up of just over two years, no developmental abnormalities were noted in the child. Ostensen et al (2004) showed that etanercept, a soluble TNF-alpha receptor fusion protein, was detected in breast milk with maximal doses noted the day following the injection. The effect on the developing immune system in the infant – if indeed the levels in breast milk are significant – remains to be seen. The European Panel on the Appropriateness of Crohn's Disease Therapy (EPACT), an international multidisciplinary panel, met recently to develop safety criteria for clinical decision-making related to the use of drugs in pregnant and nursing women with IBD. The panel was cautious about the safety of infliximab, based primarily on label **(**Mottet et al, 2007) recommendations, and emphasized the need for further large, randomized controlled trials. At the present time the risk benefit should be always assessed. Pregnant women who are receiving biologics and are in remission should continue the treatment. In the case of Infliximab and Adalumimab it should be held at 32 weeks and restarted after delivery. Breast feeding is compatible with Infliximab treatment. No data so far on breast feeding with Adalumimab although the author does not discourage breast feeding in these women.

Careful consideration should be undertaken in women of child bearing age who require biphosphonates. As with most drugs used during conception, the risk and benefits of

The role of TNF-alpha for embryonic implantation, foetal development and labour has been well studied. TNF-alpha controls arachadonic acid metabolism production through the cyclooxygenase pathway. Subsequently, TNF-alpha plays an important role in implantation and vascular permeability. TNF-alpha production is low in the first gestational trimester, but increases thereafter, reaching a peak at the onset of labour (Daher et al, 1999). These high levels have been shown to play an important role in the induction of the labour process and in delivery via augmenting uterine contractions. It has been reported that blastocyst implantation during early pregnancy may be promoted by TNF-alpha but at the same time it may mediate recurrent spontaneous abortion at a later stage of gestation. The serum levels of TNF-α and those of soluble TNFR-1 were found to be higher in women who had spontaneous early abortions (Yu et al,2005).

Anti-TNF agents include infliximab, a chimeric monoclonal IgG1 anti-TNF antibody, adalimumab, a human monoclonal IgG1 anti-TNF antibody, and etanercept, a soluble TNF receptor fusion protein linked to the Fc portion of a human IgG1. IgG1 antibody do not cross the placenta in the first trimester but cross the placenta by the late second trimester and the third trimester. Certolizumab another anti TNFα antibody, is a PEGylated Fab' fragment of humanized anti TNF alpha monoclonal antibody rather than a whole human immunoglobulin G1 (IgG1) antibody (Stephens 2006). A recent abstract on Certolizumab, reported on placental transfer. The report included 10 IBD women, with one twin delivery. There was a low level on antibodies of Certolizumab confirming the animal reports (Wolf et al, 2010; Stephens et al, 2006).

To date, there have been few comprehensive studies in IBD patients who had conceived while receiving immunobiologic therapy. Most of the publications and reports have been retrospective reviews. Roux et al (2007) reported their experience with three rheumatoid arthritis patients who became pregnant while undergoing anti-TNF-alpha therapy. Although one patient terminated her pregnancy despite no known pregnancy or foetal complications, the other two patients delivered healthy infants. Another study from the infliximab study database showed that 96 patients exposed to infliximab during pregnancy had outcomes no different than in the general population (Katz et al, 2004). In the first reported study of intentional infliximab administration for CD during pregnancy, Mahadevan et al (2005) showed, in acutely ill patients with Crohn's disease, excellent outcomes. All 10 pregnancies ended in live births and there were no congenital malformations, intrauterine growth retardation or small-for-gestational-age infants. In those ten patients, three of the infants were born prematurely and one had low birth weight (LBW).

There has not been adequate and well-controlled studies conducted in pregnant or lactating women. It has been increasingly used in autoimmune diseases such as rheumatoid arthritis, psoriatic arthritis (PsA), juvenile idiopathic arthritis (JIA) ankylosing spondylitis (AS) and in inflammatory bowel disease.

Most of the literature regarding anti-TNF-alpha therapy in pregnancy comes from studies in rheumatoid arthritis. The data have been obtained from retrospective studies, registry studies and case reports. Based on all publications no firm conclusions can be made regarding the long term safety of biologics during pregnancy. Mahadevan et al in the London position statement at the World Congress of Gastroenterogy consensus guideline (2010) considers infliximab to have a low risk and compatible with the use during conception in at least first and second trimester.

The role of TNF-alpha for embryonic implantation, foetal development and labour has been well studied. TNF-alpha controls arachadonic acid metabolism production through the cyclooxygenase pathway. Subsequently, TNF-alpha plays an important role in implantation and vascular permeability. TNF-alpha production is low in the first gestational trimester, but increases thereafter, reaching a peak at the onset of labour (Daher et al, 1999). These high levels have been shown to play an important role in the induction of the labour process and in delivery via augmenting uterine contractions. It has been reported that blastocyst implantation during early pregnancy may be promoted by TNF-alpha but at the same time it may mediate recurrent spontaneous abortion at a later stage of gestation. The serum levels of TNF-α and those of soluble TNFR-1 were found to be higher in women who had

Anti-TNF agents include infliximab, a chimeric monoclonal IgG1 anti-TNF antibody, adalimumab, a human monoclonal IgG1 anti-TNF antibody, and etanercept, a soluble TNF receptor fusion protein linked to the Fc portion of a human IgG1. IgG1 antibody do not cross the placenta in the first trimester but cross the placenta by the late second trimester and the third trimester. Certolizumab another anti TNFα antibody, is a PEGylated Fab' fragment of humanized anti TNF alpha monoclonal antibody rather than a whole human immunoglobulin G1 (IgG1) antibody (Stephens 2006). A recent abstract on Certolizumab, reported on placental transfer. The report included 10 IBD women, with one twin delivery. There was a low level on antibodies of Certolizumab confirming the animal reports (Wolf et

To date, there have been few comprehensive studies in IBD patients who had conceived while receiving immunobiologic therapy. Most of the publications and reports have been retrospective reviews. Roux et al (2007) reported their experience with three rheumatoid arthritis patients who became pregnant while undergoing anti-TNF-alpha therapy. Although one patient terminated her pregnancy despite no known pregnancy or foetal complications, the other two patients delivered healthy infants. Another study from the infliximab study database showed that 96 patients exposed to infliximab during pregnancy had outcomes no different than in the general population (Katz et al, 2004). In the first reported study of intentional infliximab administration for CD during pregnancy, Mahadevan et al (2005) showed, in acutely ill patients with Crohn's disease, excellent outcomes. All 10 pregnancies ended in live births and there were no congenital malformations, intrauterine growth retardation or small-for-gestational-age infants. In those ten patients, three of the infants were born prematurely and one had low birth weight

There has not been adequate and well-controlled studies conducted in pregnant or lactating women. It has been increasingly used in autoimmune diseases such as rheumatoid arthritis, psoriatic arthritis (PsA), juvenile idiopathic arthritis (JIA) ankylosing spondylitis (AS) and in

Most of the literature regarding anti-TNF-alpha therapy in pregnancy comes from studies in rheumatoid arthritis. The data have been obtained from retrospective studies, registry studies and case reports. Based on all publications no firm conclusions can be made regarding the long term safety of biologics during pregnancy. Mahadevan et al in the London position statement at the World Congress of Gastroenterogy consensus guideline (2010) considers infliximab to have a low risk and compatible with the use during

spontaneous early abortions (Yu et al,2005).

al, 2010; Stephens et al, 2006).

inflammatory bowel disease.

conception in at least first and second trimester.

(LBW).

Recently, the mother risk program in Canada published a review of 300 pregnancies in women who received anti TNF alpha during conception. The review suggested that infliximab carries low foetal risk and is compatible with use during conception and the first two trimesters of pregnancy (Djokanovic et al, 2011).

A recent observational study by the Leuven, group in Belgium, was published. They assessed pregnancy outcomes in 212 women with IBD. Pregnancy outcome was assessed in 42 pregnancies in women who received anti-TNF treatment (35 IFX, 7 ADA) and were compared with that in 23 pregnancies prior to IBD diagnosis, 78 pregnancies before start of IFX, 53 pregnancies with indirect exposure to IFX, and 56 matched pregnancies in healthy women. Their conclusion was that direct exposure to anti-TNF treatment during pregnancy was not related to a higher incidence of adverse outcomes than IBD overall (Schnitzler et al, 2011).

Vasiliauskas monitored a pregnant patient who continued on standard-dose infliximab therapy during her pregnancy and lactation. The measurement of serum infliximab in the breastfed infant was 39.5 μg/mL or less and the drug was not detected in the breast milk. Importantly, despite continued breast-feeding and adherence to therapy, the levels declined in the infant over the subsequent six months. They concluded that infliximab levels were likely due to placental transfer and less likely the result of breastfeeding (Vasiliauskas et al, 2006). The blood levels in the newborn were within the therapeutic range. The authors recommended that such therapy be avoided after 30 weeks gestation when possible. As of the date of this publication, no increased risk of embryotoxicity, teratogenicity or adverse pregnancy outcome has been reported in patients treated with anti-TNF therapy (Skomsvoll et al, 2007).

The majority of studies regarding breast feeding while receiving anti-TNF therapy has not demonstrated any adverse outcomes. The data, however, appear limited. In a case report publication in which serial sampling of breast milk over a one-month time period failed to detect any concentrations of infliximab. Furthermore, at a follow-up of just over two years, no developmental abnormalities were noted in the child. Ostensen et al (2004) showed that etanercept, a soluble TNF-alpha receptor fusion protein, was detected in breast milk with maximal doses noted the day following the injection. The effect on the developing immune system in the infant – if indeed the levels in breast milk are significant – remains to be seen. The European Panel on the Appropriateness of Crohn's Disease Therapy (EPACT), an international multidisciplinary panel, met recently to develop safety criteria for clinical decision-making related to the use of drugs in pregnant and nursing women with IBD. The panel was cautious about the safety of infliximab, based primarily on label **(**Mottet et al, 2007) recommendations, and emphasized the need for further large, randomized controlled trials.

At the present time the risk benefit should be always assessed. Pregnant women who are receiving biologics and are in remission should continue the treatment. In the case of Infliximab and Adalumimab it should be held at 32 weeks and restarted after delivery. Breast feeding is compatible with Infliximab treatment. No data so far on breast feeding with Adalumimab although the author does not discourage breast feeding in these women.

## **20. Bisphosphonates (FDA Class C)**

Careful consideration should be undertaken in women of child bearing age who require biphosphonates. As with most drugs used during conception, the risk and benefits of biphosphonates should be carefully weighed in these women.

Approach to the Management of the Pregnant

facial clefts. CMAJ, 2011,April11.

Inflamm Bowel Dis. 2009 Jan; 15(1):25-8.

Gastroenterol. 1995;90:1918–1922.

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(suppl 1):i36-i58.

J Matern Fetal Neonatal Med. 2004; 15:237–41.

anomalies. Am. J. Med. Genet. 1999; 86(3): 242–244

Pregnant Women Fetal Diagn Ther 1998; 13:136-140

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Chakrabarty, G \*, A Poullis Gut 2011; 60:A135 doi:10.1136/gut.2011.239301.287

127:264–267.

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Anders Hviid, Ditte Mølgaard-Nielsen. Corticosteroid use during pregnancy and risk of oro

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Cantu JM, Garcia-Cruz D. Midline facial defect as a teratogenic effect of metronidazole.

Caprilli R, Gassul MA, Escher JC, Moser G, et al. European evidence based consensus on the

Carmichael SL, Shaw GM. Maternal corticosteroid use and risk of selected congenital

Carter MJ, Lobo AJ, Travis SPL., IBD Section, British Society of Gastroenterology Guidelines for the management of inflammatory bowel disease in adults. Gut. 2004; 53:V1–16. Caruso A, De Carolis S, Sergio Ferrazzani, Carmen Trivellini, Carmen Mastromarino, Mauro

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Kuhlmann RS, Otterson MF, Emmons J, Knox J, Binion DG. / Budesonide induction and maintenance therapy for Crohn's disease during pregnancy.

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Alstead EM. Inflammatory bowel disease in pregnancy. Postgrad Med J. 2002; 78:23–26. Alstead EM, Nelson-Piercy C. Inflammatory bowel disease in pregnancy. Gut. 2161.

disease. Gastroenterol. Clin. North Am. 1999;28(2):255–281, vii.

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There are several biphosphonates currently approved for use in osteoporosis including: alendronate (Fosamax ®), etidronate (Didrocal ®), risedronate (Actonel ®) and zoledronic acid (Aclasta®).

Biphosphonates are used to treat and to prevent corticosteroids induced osteoporosis. Approximately 50% of the Biphosphonates binds to the skeleton and the rest is excreted non-metabolized by the kidneys (Papapoulos, 2008). The long-term effects of alendronate, one of the Biphosphonates, on human bone development are unknown and the half-life of alendronate is greater than 10 years. The concern with long-term biphosphonates treatment is that the drug is slowly released from maternal bone and may result in continuous lowlevel exposure to the foetus during gestation. The released bisphosphonate can cross the placenta and incorporate into foetal bone. In animal studies where biphosphonates were given to pregnant rats, the drug appears to cross the placenta, accumulate in the foetal skeleton, decrease foetal weight, decrease bone growth, and lead to protracted deliveries and neonatal deaths (Patlas et al, 1999).

## **21. Summary and conclusion**

Managing a pregnant woman with inflammatory bowel disease is challenging but often rewarding. This management should include the patient and her partner, in association with the family physician, the gastroenterologist and the obstetrician. Most women with IBD, who are in remission, are as likely as women without IBD to conceive and have a normal pregnancy. These patients should be encouraged to continue the medications throughout the pregnancy and while breast feeding.

Women with ulcerative colitis who have undergone ileal pouch-anal anastomosis operation are less likely to conceive. Women with active ulcerative colitis who require colectomy, and are planning to have a family, should reconsider having a pouch surgery. They should have an ileostomy and postpone a pouch until after conceiving. With the exception of Methotrexate, most drugs used in IBD are relatively safe to administer in pregnancy and can be used while breast feeding.

The newer biologic drugs appear to be quite effective and safe during pregnancy and with breastfeeding. Since these drugs cross the placenta mostly in the third trimester they should not be administered after 32 week of gestation.

Despite all our knowledge and availability of literature the risk benefit should be always weighed. The decision of the mother should always be respected, should she decide on not taking the medications.

#### **Take home messages:**


## **22. References**

192 Inflammatory Bowel Disease – Advances in Pathogenesis and Management

There are several biphosphonates currently approved for use in osteoporosis including: alendronate (Fosamax ®), etidronate (Didrocal ®), risedronate (Actonel ®) and zoledronic

Biphosphonates are used to treat and to prevent corticosteroids induced osteoporosis. Approximately 50% of the Biphosphonates binds to the skeleton and the rest is excreted non-metabolized by the kidneys (Papapoulos, 2008). The long-term effects of alendronate, one of the Biphosphonates, on human bone development are unknown and the half-life of alendronate is greater than 10 years. The concern with long-term biphosphonates treatment is that the drug is slowly released from maternal bone and may result in continuous lowlevel exposure to the foetus during gestation. The released bisphosphonate can cross the placenta and incorporate into foetal bone. In animal studies where biphosphonates were given to pregnant rats, the drug appears to cross the placenta, accumulate in the foetal skeleton, decrease foetal weight, decrease bone growth, and lead to protracted deliveries

Managing a pregnant woman with inflammatory bowel disease is challenging but often rewarding. This management should include the patient and her partner, in association with the family physician, the gastroenterologist and the obstetrician. Most women with IBD, who are in remission, are as likely as women without IBD to conceive and have a normal pregnancy. These patients should be encouraged to continue the medications throughout

Women with ulcerative colitis who have undergone ileal pouch-anal anastomosis operation are less likely to conceive. Women with active ulcerative colitis who require colectomy, and are planning to have a family, should reconsider having a pouch surgery. They should have an ileostomy and postpone a pouch until after conceiving. With the exception of Methotrexate, most drugs used in IBD are relatively safe to administer in pregnancy and can

The newer biologic drugs appear to be quite effective and safe during pregnancy and with breastfeeding. Since these drugs cross the placenta mostly in the third trimester they should

Despite all our knowledge and availability of literature the risk benefit should be always weighed. The decision of the mother should always be respected, should she decide on not

1. Patient education is of primary importance in a successful outcome in IBD women

2. IBD patients in remission have the same chance in getting pregnant as non IBD

3. Pregnant IBD patients outcomes are better if patients are maintained in remission.

7. Breast feeding should be encouraged in women receiving most of IBD drugs.

acid (Aclasta®).

and neonatal deaths (Patlas et al, 1999).

the pregnancy and while breast feeding.

not be administered after 32 week of gestation.

4. Ileoanal pouch patients have a lower rate of conception.

5. Biologics drugs appear to be safe during pregnancy and in lactation. 6. Biologic drugs, if possible, should be withheld at 32 weeks gestation

be used while breast feeding.

taking the medications. **Take home messages:** 

during pregnancy.

patients.

**21. Summary and conclusion** 

Alstead EM. Inflammatory bowel disease in pregnancy. Postgrad Med J. 2002; 78:23–26.

Alstead EM, Nelson-Piercy C. Inflammatory bowel disease in pregnancy. Gut. 2161.


Approach to the Management of the Pregnant

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**11** 

*Croatia* 

**Bone Morphogenetic Proteins and Signaling** 

Inflammatory bowel disease (IBD) is a chronic, relapsing disease of the gastrointestinal (GI) tract of uncertain origin. Its two main phenotypes are Crohn's disease (CD) and ulcerative colitis (UC). CD affects any part of the GI tract and is characterized by transmural inflammation, whereas UC is confined to the colon and affects only the mucosal layer. IBD is thought to occur in genetically predisposed individuals that develop an abnormal immune response to enteric bacteria in the intestinal mucosa (Podolsky, 2002; Xavier RJ & Podolsky, 2007). Disease occurs as a result of complex and dynamic interactions between immune and non-immune cells as well as the cross-talk between intestinal epithelium and mesenchyme (Danese, 2011; MacDonald et al., 2011; Strober & Fuss, 2011). Therefore, factors that are able to influence both interactions may be very

Bone morphogenetic proteins (BMPs) are a large group of structurally related proteins that belong to the transforming growth factor-β (TGF-β) superfamily. Along with their primarily osteogenic function their importance in development, proliferation and morphogenesis of a variety of cells and tissues has been shown (Hogan, 1996; Vukicevic et al., 1989; 1995; Wozney et al., 1988). In addition, association of BMPs with healing processes of different non-skeletal tissues and organs was also described (Lories et al., 2005; Martinovic et al., 2002; Nguyen et al., 2008; Simic & Vukicevic, 2004; Turk et al., 2009; Vukicevic et al., 1996; Vukicevic & Grgurevic, 2009). Due to their wide-range of effects, they are commonly named "body morphogenetic proteins" (Reddi, 2005). Perturbations in BMP expression and BMP signaling pathway have been associated with the pathological conditions linked to several human diseases such as inflammatory bowel disease (IBD) (Allaire et al., 2011; Burke et al.,

In this chapter we will discuss the importance of BMPs in gut development and hereditary diseases as well as their influence on cellular and molecular events that occur in IBD and fibrogenesis, the most common complication of IBD. Furthermore, we will address the therapeutical potential of BMPs, especially BMP7 in treatment of IBD. Finally, we will explore the possibility of BMP pathway components as putative biomarkers of gut tumor

important for the pathogenesis and treatment of IBD.

**1. Introduction** 

2007; Krishnan et al., 2011).

development and progression.

**Pathway in Inflammatory Bowel Disease**

Ivana Maric1, Tamara Turk Wensveen2,Ivana Smoljan3,

*1Department of Anatomy, Faculty of Medicine, University of Rijeka* 

Zeljka Crncevic Orlic2 and Dragica Bobinac1

*3Psychiatric Hospital Rab* 

*2Department of Internal Medicine, Clinical Hospital Rijeka* 


## **Bone Morphogenetic Proteins and Signaling Pathway in Inflammatory Bowel Disease**

Ivana Maric1, Tamara Turk Wensveen2,Ivana Smoljan3, Zeljka Crncevic Orlic2 and Dragica Bobinac1 *1Department of Anatomy, Faculty of Medicine, University of Rijeka 2Department of Internal Medicine, Clinical Hospital Rijeka 3Psychiatric Hospital Rab Croatia* 

## **1. Introduction**

198 Inflammatory Bowel Disease – Advances in Pathogenesis and Management

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Inflammatory bowel disease (IBD) is a chronic, relapsing disease of the gastrointestinal (GI) tract of uncertain origin. Its two main phenotypes are Crohn's disease (CD) and ulcerative colitis (UC). CD affects any part of the GI tract and is characterized by transmural inflammation, whereas UC is confined to the colon and affects only the mucosal layer. IBD is thought to occur in genetically predisposed individuals that develop an abnormal immune response to enteric bacteria in the intestinal mucosa (Podolsky, 2002; Xavier RJ & Podolsky, 2007). Disease occurs as a result of complex and dynamic interactions between immune and non-immune cells as well as the cross-talk between intestinal epithelium and mesenchyme (Danese, 2011; MacDonald et al., 2011; Strober & Fuss, 2011). Therefore, factors that are able to influence both interactions may be very important for the pathogenesis and treatment of IBD.

Bone morphogenetic proteins (BMPs) are a large group of structurally related proteins that belong to the transforming growth factor-β (TGF-β) superfamily. Along with their primarily osteogenic function their importance in development, proliferation and morphogenesis of a variety of cells and tissues has been shown (Hogan, 1996; Vukicevic et al., 1989; 1995; Wozney et al., 1988). In addition, association of BMPs with healing processes of different non-skeletal tissues and organs was also described (Lories et al., 2005; Martinovic et al., 2002; Nguyen et al., 2008; Simic & Vukicevic, 2004; Turk et al., 2009; Vukicevic et al., 1996; Vukicevic & Grgurevic, 2009). Due to their wide-range of effects, they are commonly named "body morphogenetic proteins" (Reddi, 2005). Perturbations in BMP expression and BMP signaling pathway have been associated with the pathological conditions linked to several human diseases such as inflammatory bowel disease (IBD) (Allaire et al., 2011; Burke et al., 2007; Krishnan et al., 2011).

In this chapter we will discuss the importance of BMPs in gut development and hereditary diseases as well as their influence on cellular and molecular events that occur in IBD and fibrogenesis, the most common complication of IBD. Furthermore, we will address the therapeutical potential of BMPs, especially BMP7 in treatment of IBD. Finally, we will explore the possibility of BMP pathway components as putative biomarkers of gut tumor development and progression.

Bone Morphogenetic Proteins and Signaling Pathway in Inflammatory Bowel Disease 201

heteromeric complexes and activate downstream signaling molecules through Smad and non-Smad signaling pathway (Fig. 3) (Korchynskyi & ten Dijke, 2002; Massague et al., 2005; Miyazono et al., 2005). Both receptors are required to activate the signaling pathway. By BMP ligand binding, type II receptor phosphorylates the type I receptor which propagates

(a) BMP-receptors (b) BR-Smad and AR-Smad proteins

The intracellular mediators of BMP signaling transduction are Smad transcription factors. Smad is named after human Sma1 assessment and its identification with similar genes found in *Drosophila* (Drosophile mothers against dpp – Mad) and *Caenorhabditis elegans* (Sma) (Liu et al., 1996). Three subclasses of Smad proteins have been described based on their structure and function: receptor regulated Smads (R-Smads), common mediator Smad (co-Smad), and inhibitory Smads (I-Smads) (Fig. 2). R-Smads are further subdivided into BR-Smads referred to as BMP signaling (Smad1/5/8) and AR-Smads referred to as activin/TGFβ signaling (Smad2/3). They are phyosphorylated by the type I receptors, interact with co-Smad (Smad4) and translocate into the nucleus initiating transcription of BMP response genes (Kawabata et al., 1998; Miyazono et al., 2010). Inhibitory Smads (I-Smad6/7) compete with R-Smads for the activated type I receptor binding site. Smad7 inhibits both activin/TGFβ and BMP signaling pathway while Smad6 preferentially inhibits

BMP signaling is controlled also by soluble BMP antagonists. They exert their function by direct binding of BMPs and prevent functional receptor/ligand interaction (Lein et al., 2002; Piccolo et al., 1996; Yanagita, 2005; Zimmerman et al., 1995). Several structurally distinct classes of inhibitory BMP binding proteins have been described in vertebrates: twisted gastrulation (TSG), chordin and noggin and the DAN-family of inhibitors. The DAN family includes a large number of members such as DAN, cerebrus, protein related to DAN and cerebrus (PRDC), gremlin, Cer1, USAG-1 and sclerostin (Bouwmeester et al., 1996; Hsu et al., 1998; Stanley et al., 1998). They bind different BMPs with various degrees of affinity and specifity. For instance, noggin and chordin bind BMP2 and -4 with higher affinity than BMP7 whereas sclerostin binds with greater affinity BMP6 and -7 (Lein et al., 2002; Yanagita, 2005).

**3. BMPs and their signaling pathway in gastrointestinal tract** 

**3.1 BMPs and their signaling pathway in gastrointestinal tract development** 

BMPs and their signaling pathways are important for the normal development of the gastrointestinal tract. They are active from the earliest stage of development and revealed to be essential for the intestinal growth and morphogenesis. It has also been shown that BMP

the signal (Massague et al., 2005, Wrana et al., 1994).

Fig. 2. Components of TGFβ/BMP signaling pathway

BMP signaling (Hanyu et al., 2001).

## **2. Bone morphogenetic proteins**

BMPs comprise a group of very important signaling molecules, which is demonstrated by the the fact that several of these proteins, as well as their intracellular signaling components have been conserved in *Drosophila* and *Caenorhabditis elegans*. They are originally isolated from bone and major contribution to their isolation and characterization was made by Sampath and Reddi (Sampath & Reddi, 1981; Sampath et al., 1987). BMPs are a large family within the transforming growth factor β (TGFβ) superfamily. Twenty BMP family members have been isolated and characterized so far. BMPs are divided into 4 groups based on their structure and function: the BMP2/4 group consists of BMP2, BMP4 and Decapentaplegic (dpp) in Drosophila, the OP-1 group is made up of BMP5, BMP6, BMP7 or osteogenic protein (OP1), BMP8 (OP2) and 60A in Drosophila, the GDF5 group includes the growthdifferentiation factor-5 (GDF5) or cartilage-derived morphogenetic protein-1 (CDMP1), GDF6 (CDMP2 or BMP13) and GDF7 (BMP12), and finally the forth group includes BMP9 and BMP10 (Miyazono, 2000; Miyazono et al., 2010). Grouping of BMPs is continually subject to changes due to studies which reveal their structure.

Fig. 1. TGFβ superfamily (according to Celeste et al., 1990).

BMPs are 30-38 kDa dimeric molecules. Their structure is very important for biological activity in vivo. They are synthesized as large precursor proteins in the cytoplasm that are proteolytically processed to yield mature proteins (Kingsley, 1994). Mature BMP molecules are characterized by the formation of a cysteine knot with the seven conserved cysteine domains. Active form of the molecule is dimeric as a homodimer or heterodimer (Rueger, 2002).

BMPs transduce their signals by binding to two different transmembrane serine/threonine kinase receptors, type I (BMPRI) and type II (BMPRII). Three type I and three type II receptors are identified for BMP ligand binding (Fig. 2). Type I receptors include activin receptor type IA (ActRIA or ALK2) and BMP receptors type IA and IB (BRIA or ALK3; BRIB or ALK6), while type II receptors are BMP receptor type II (BRII), activin receptor type IIA and IIB (ActRIIA and ActRIIB) (Miyazono et al., 2005, Sieber et al., 2009). Receptors form heteromeric complexes and activate downstream signaling molecules through Smad and non-Smad signaling pathway (Fig. 3) (Korchynskyi & ten Dijke, 2002; Massague et al., 2005; Miyazono et al., 2005). Both receptors are required to activate the signaling pathway. By BMP ligand binding, type II receptor phosphorylates the type I receptor which propagates the signal (Massague et al., 2005, Wrana et al., 1994).

200 Inflammatory Bowel Disease – Advances in Pathogenesis and Management

BMPs comprise a group of very important signaling molecules, which is demonstrated by the the fact that several of these proteins, as well as their intracellular signaling components have been conserved in *Drosophila* and *Caenorhabditis elegans*. They are originally isolated from bone and major contribution to their isolation and characterization was made by Sampath and Reddi (Sampath & Reddi, 1981; Sampath et al., 1987). BMPs are a large family within the transforming growth factor β (TGFβ) superfamily. Twenty BMP family members have been isolated and characterized so far. BMPs are divided into 4 groups based on their structure and function: the BMP2/4 group consists of BMP2, BMP4 and Decapentaplegic (dpp) in Drosophila, the OP-1 group is made up of BMP5, BMP6, BMP7 or osteogenic protein (OP1), BMP8 (OP2) and 60A in Drosophila, the GDF5 group includes the growthdifferentiation factor-5 (GDF5) or cartilage-derived morphogenetic protein-1 (CDMP1), GDF6 (CDMP2 or BMP13) and GDF7 (BMP12), and finally the forth group includes BMP9 and BMP10 (Miyazono, 2000; Miyazono et al., 2010). Grouping of BMPs is continually

**2. Bone morphogenetic proteins** 

subject to changes due to studies which reveal their structure.

Fig. 1. TGFβ superfamily (according to Celeste et al., 1990).

(Rueger, 2002).

BMPs are 30-38 kDa dimeric molecules. Their structure is very important for biological activity in vivo. They are synthesized as large precursor proteins in the cytoplasm that are proteolytically processed to yield mature proteins (Kingsley, 1994). Mature BMP molecules are characterized by the formation of a cysteine knot with the seven conserved cysteine domains. Active form of the molecule is dimeric as a homodimer or heterodimer

BMPs transduce their signals by binding to two different transmembrane serine/threonine kinase receptors, type I (BMPRI) and type II (BMPRII). Three type I and three type II receptors are identified for BMP ligand binding (Fig. 2). Type I receptors include activin receptor type IA (ActRIA or ALK2) and BMP receptors type IA and IB (BRIA or ALK3; BRIB or ALK6), while type II receptors are BMP receptor type II (BRII), activin receptor type IIA and IIB (ActRIIA and ActRIIB) (Miyazono et al., 2005, Sieber et al., 2009). Receptors form The intracellular mediators of BMP signaling transduction are Smad transcription factors. Smad is named after human Sma1 assessment and its identification with similar genes found in *Drosophila* (Drosophile mothers against dpp – Mad) and *Caenorhabditis elegans* (Sma) (Liu et al., 1996). Three subclasses of Smad proteins have been described based on their structure and function: receptor regulated Smads (R-Smads), common mediator Smad (co-Smad), and inhibitory Smads (I-Smads) (Fig. 2). R-Smads are further subdivided into BR-Smads referred to as BMP signaling (Smad1/5/8) and AR-Smads referred to as activin/TGFβ signaling (Smad2/3). They are phyosphorylated by the type I receptors, interact with co-Smad (Smad4) and translocate into the nucleus initiating transcription of BMP response genes (Kawabata et al., 1998; Miyazono et al., 2010). Inhibitory Smads (I-Smad6/7) compete with R-Smads for the activated type I receptor binding site. Smad7 inhibits both activin/TGFβ and BMP signaling pathway while Smad6 preferentially inhibits BMP signaling (Hanyu et al., 2001).

BMP signaling is controlled also by soluble BMP antagonists. They exert their function by direct binding of BMPs and prevent functional receptor/ligand interaction (Lein et al., 2002; Piccolo et al., 1996; Yanagita, 2005; Zimmerman et al., 1995). Several structurally distinct classes of inhibitory BMP binding proteins have been described in vertebrates: twisted gastrulation (TSG), chordin and noggin and the DAN-family of inhibitors. The DAN family includes a large number of members such as DAN, cerebrus, protein related to DAN and cerebrus (PRDC), gremlin, Cer1, USAG-1 and sclerostin (Bouwmeester et al., 1996; Hsu et al., 1998; Stanley et al., 1998). They bind different BMPs with various degrees of affinity and specifity. For instance, noggin and chordin bind BMP2 and -4 with higher affinity than BMP7 whereas sclerostin binds with greater affinity BMP6 and -7 (Lein et al., 2002; Yanagita, 2005).

## **3. BMPs and their signaling pathway in gastrointestinal tract**

## **3.1 BMPs and their signaling pathway in gastrointestinal tract development**

BMPs and their signaling pathways are important for the normal development of the gastrointestinal tract. They are active from the earliest stage of development and revealed to be essential for the intestinal growth and morphogenesis. It has also been shown that BMP

Bone Morphogenetic Proteins and Signaling Pathway in Inflammatory Bowel Disease 203

signaling activity is detected also in the endoderm, future epithelium with prominent delay of BMP signaling activation in the colon. Inhibition of BMP activation by Bapx1 misexpression in the chick gut mesoderm results in diminished influence of mesoderm to endoderm-ectoderm signaling. This leads to an altered gut phenotype with marked muscular hypertrophy followed by abnormality in the ENS and epithelium. Decreased BMP signaling in mice overexpressing the BMP-antagonist noggin leads to abnormal villus morphogenesis of mouse proximal intestine. This was associated with stromal and epithelial hyperplasia, and ectopic crypt formation due to low levels of BmprIA and pSmad1/5/8 (Batts et al., 2006). A similar abnormal phenotype was found in the large intestine. These results suggest that BMP signaling restricts the site of crypt formation to the intervillus region and normally suppresses crypt formation in the villus. Human GI diseases which are accompanied by abnormal intestinal morphology, like JP or ENS disorder and even more chronic intestinal diseases associated with muscular hypertrophy such as Hirschprung's disease, could be associated

with defects in the BMP signaling pathway (Amiel & Lyonnet, 2001; He et al., 2004).

BMPs and their signaling pathway are expressed in normal intestine and colon. It was shown that BMP2, the BMP receptors (Ia, Ib, II), phosphorylated Smad1 and Smad4 are present in mature colonocytes at the epithelial surface of normal human and mouse colon (Hardwick et al., 2004). The expression of BMP receptors was also found in colonic epithelial cell lines and BMP2 treatment in vitro resulted in inhibition of proliferation and induction of apoptosis. BMPRIA and BMP2 are highly expressed in the villus epithelium and distal/surface epithelium of the adult mouse small and large intestine with increase expression in stroma and crypt epithelium (Batts et al., 2006; Haramis et al., 2004). BMP4 is expressed in stromal cells and mesenchimal cells surrounding the crypt and glands of the

The expression profile of genes in the mouse intestine showed a difference between the expression of BMP signaling components in the epithelium and mesenchyme (Li et al., 2007). BMP signaling is observed in both epithelial and mesenchymal compartments, though studies to date have primarily addressed epithelial signal transduction (He et al., 2004). BMP2, BMP4, BMP5, BMP6 and BMPRII are expressed in mesenchymal compartments while only BMP7 showed epithelial enrichment. BMP7 was present in surface epithelial cells and crypts of the normal colon mucosa (Grijelmo et al., 2007). BMP receptors type I (BMPRIA or ALK3 and Acvr1 or ALK2) are present in mesenchymal compartments while the expression of BMPRIB (ALK6) was found in both compartments. Smads are expressed in both epithelial and mesenchymal compartments with slight enrichments in the mesenchyme while Smad4 expression was approximately equal in both compartments. Along the colon crypt, a different pattern of Smad4 expression was observed with high

Apart from noggin, the BMP pathway inhibitors are located exclusively in the mesenchyme. The gene expression analysis of normal human colon tops and basal crypts revealed difference in nine hundred and sixty-nine cDNA clones from these two compartments (Kosinski et al., 2007). BMP1, BMP2, BMP5, BMP7, SMAD7 and BMPRII were highly expressed in colon tops while expression of the BMP antagonists gremlin 1, gremlin 2 and chordin-like 1 was found in basal colon crypts originating from myofibroblasts and smooth muscle cells (Fig. 4). It was also shown that activation of extracellular calcium-sensing

**3.2 BMPs and their signaling pathway in the normal gastrointestinal tract** 

small and large intestine (Haramis et al., 2004; He et al., 2004).

expression in the zone of terminal differentiation (Korchynskyi et al., 1999).

Fig. 3. BMP signaling pathway (with kind permission from Springer Science+Business Media: Bone morphogenetic proteins: from laboratory, to clinical practice, Bone morphogentic protein receptors and their nuclear effectors in bone formation, 2002, pp. 31– 60, Korchynskyi, O. & ten Dijke, P. Fig. 3)

signaling is a mediator in epithelial and mesenchymal stroma interaction which is required for the intestinal growth, morphogenesis, differentiation and homeostasis (Batts et al., 2006; de Santa Barbara et al., 2005). Initial investigation of the BMP expression in the GI tract showed limited expression of individual BMP and its receptors during development. BMP6 and BMP7 were found in the developing human and mouse intestine limiting its expression to the smooth muscle cells and intestinal epithelium, respectively (Helder et al., 1995; Perr et al., 1999). Expression of BMP type I receptor (ALK6) was found in the stomach and its pyloric region (Dewulf et al.,1995). It has also been noticed that villus formation depends on high BMP2 and -4 expressions in the condensed mesenchyme underlying sites of future villus formation and may inhibit crypt formation in the overlying epithelium (Karlsson et al., 2000). In addition, BMP2 and its receptors (IB and II) were expressed in the smooth muscle progenitors of mouse embryonic GI tract with evident BMP2-induced smooth muscle differentiation and phenotype (Goldman et al., 2009).

De Santa Barbara et al. (2005) noticed that the BMP signaling pathway showed wider expression than BMP ligands in the chick developing gut. This pathway is activated in all three tissue layers of the GI tract, allowing interaction and reciprocal communication. The BMP signaling activity was found in the mesoderm during the differentiation into visceral smooth muscle and was downregulated by the completion of this process. A similar observation was made for the enteric nervous system (ENS) during its derivation of the ectoderm. BMP

Fig. 3. BMP signaling pathway (with kind permission from Springer Science+Business Media: Bone morphogenetic proteins: from laboratory, to clinical practice, Bone

60, Korchynskyi, O. & ten Dijke, P. Fig. 3)

muscle differentiation and phenotype (Goldman et al., 2009).

morphogentic protein receptors and their nuclear effectors in bone formation, 2002, pp. 31–

signaling is a mediator in epithelial and mesenchymal stroma interaction which is required for the intestinal growth, morphogenesis, differentiation and homeostasis (Batts et al., 2006; de Santa Barbara et al., 2005). Initial investigation of the BMP expression in the GI tract showed limited expression of individual BMP and its receptors during development. BMP6 and BMP7 were found in the developing human and mouse intestine limiting its expression to the smooth muscle cells and intestinal epithelium, respectively (Helder et al., 1995; Perr et al., 1999). Expression of BMP type I receptor (ALK6) was found in the stomach and its pyloric region (Dewulf et al.,1995). It has also been noticed that villus formation depends on high BMP2 and -4 expressions in the condensed mesenchyme underlying sites of future villus formation and may inhibit crypt formation in the overlying epithelium (Karlsson et al., 2000). In addition, BMP2 and its receptors (IB and II) were expressed in the smooth muscle progenitors of mouse embryonic GI tract with evident BMP2-induced smooth

De Santa Barbara et al. (2005) noticed that the BMP signaling pathway showed wider expression than BMP ligands in the chick developing gut. This pathway is activated in all three tissue layers of the GI tract, allowing interaction and reciprocal communication. The BMP signaling activity was found in the mesoderm during the differentiation into visceral smooth muscle and was downregulated by the completion of this process. A similar observation was made for the enteric nervous system (ENS) during its derivation of the ectoderm. BMP signaling activity is detected also in the endoderm, future epithelium with prominent delay of BMP signaling activation in the colon. Inhibition of BMP activation by Bapx1 misexpression in the chick gut mesoderm results in diminished influence of mesoderm to endoderm-ectoderm signaling. This leads to an altered gut phenotype with marked muscular hypertrophy followed by abnormality in the ENS and epithelium. Decreased BMP signaling in mice overexpressing the BMP-antagonist noggin leads to abnormal villus morphogenesis of mouse proximal intestine. This was associated with stromal and epithelial hyperplasia, and ectopic crypt formation due to low levels of BmprIA and pSmad1/5/8 (Batts et al., 2006). A similar abnormal phenotype was found in the large intestine. These results suggest that BMP signaling restricts the site of crypt formation to the intervillus region and normally suppresses crypt formation in the villus. Human GI diseases which are accompanied by abnormal intestinal morphology, like JP or ENS disorder and even more chronic intestinal diseases associated with muscular hypertrophy such as Hirschprung's disease, could be associated with defects in the BMP signaling pathway (Amiel & Lyonnet, 2001; He et al., 2004).

#### **3.2 BMPs and their signaling pathway in the normal gastrointestinal tract**

BMPs and their signaling pathway are expressed in normal intestine and colon. It was shown that BMP2, the BMP receptors (Ia, Ib, II), phosphorylated Smad1 and Smad4 are present in mature colonocytes at the epithelial surface of normal human and mouse colon (Hardwick et al., 2004). The expression of BMP receptors was also found in colonic epithelial cell lines and BMP2 treatment in vitro resulted in inhibition of proliferation and induction of apoptosis. BMPRIA and BMP2 are highly expressed in the villus epithelium and distal/surface epithelium of the adult mouse small and large intestine with increase expression in stroma and crypt epithelium (Batts et al., 2006; Haramis et al., 2004). BMP4 is expressed in stromal cells and mesenchimal cells surrounding the crypt and glands of the small and large intestine (Haramis et al., 2004; He et al., 2004).

The expression profile of genes in the mouse intestine showed a difference between the expression of BMP signaling components in the epithelium and mesenchyme (Li et al., 2007). BMP signaling is observed in both epithelial and mesenchymal compartments, though studies to date have primarily addressed epithelial signal transduction (He et al., 2004). BMP2, BMP4, BMP5, BMP6 and BMPRII are expressed in mesenchymal compartments while only BMP7 showed epithelial enrichment. BMP7 was present in surface epithelial cells and crypts of the normal colon mucosa (Grijelmo et al., 2007). BMP receptors type I (BMPRIA or ALK3 and Acvr1 or ALK2) are present in mesenchymal compartments while the expression of BMPRIB (ALK6) was found in both compartments. Smads are expressed in both epithelial and mesenchymal compartments with slight enrichments in the mesenchyme while Smad4 expression was approximately equal in both compartments. Along the colon crypt, a different pattern of Smad4 expression was observed with high expression in the zone of terminal differentiation (Korchynskyi et al., 1999).

Apart from noggin, the BMP pathway inhibitors are located exclusively in the mesenchyme. The gene expression analysis of normal human colon tops and basal crypts revealed difference in nine hundred and sixty-nine cDNA clones from these two compartments (Kosinski et al., 2007). BMP1, BMP2, BMP5, BMP7, SMAD7 and BMPRII were highly expressed in colon tops while expression of the BMP antagonists gremlin 1, gremlin 2 and chordin-like 1 was found in basal colon crypts originating from myofibroblasts and smooth muscle cells (Fig. 4). It was also shown that activation of extracellular calcium-sensing

Bone Morphogenetic Proteins and Signaling Pathway in Inflammatory Bowel Disease 205

cytokine production (Dieleman et al., 1994; Kitajima et al., 1999). Trinitrobenzene sulfonic acid (TNBS) dissolved in ethanol induces a CD-like colitis, with transmural inflammation (Elson et al., 1995). Ethanol is thought to cause a transient increase in intestinal permeability, allowing TNBS to invade the musocal layers and act as a hapten to form complete antigens with tissue proteins. This provokes an intense and sustained inflammatory response and breaks T-cell tolerance to mucosal antigens (Boismenu & Chen, 2000). Genetic colitis models such as gene knockout and transgene models offer another approach in experimental IBD research. They imply genetic modifications that favor the development of colitis. IL-2 deficient mice spontaneously develop a UC-like colitis in 100% of cases, while mice lacking the IL-10 gene develop chronic colitis only in the presence of enteric bacteria (Kuhn et al., 1993; Sadlack et al., 1993). Other genetic models include T cell receptor, STAT3 and IL-17 gene manipulation

Monteleone et al. (2001) were among the first groups to show the disturbance of the TGF-β1 signaling pathway in human IBD. They found that Smad7, an inhibitor of BMP-signaling, was overexpressed, whereas Smad3, an R-Smad of this signaling pathway, was downregulated. Due to Smad7 inhibition, lamina propria mononuclear cells (LPMC) isolated from mucosa of CD patients were not able to respond to TGF-β1 and to downregulate proinflammatory cytokine expression, especially of TNFα and IFNγ. Moreover, Smad7 inhibition by specific antisense oligonucleotides allowed LPMC to respond to TGF-β1 restoring TGF-β1 signaling by increasing pSmad3 and decreasing Smad7, and provide TGF-β1-mediated inhibition of proinflammatory cytokine production in experimentally induced colitis (Boirivant et al., 2006). Previously, it was shown that mice with targeted disruption of the TGF-β1 gene or one of its intracellular signaling components develop multifocal inflammation (Shull et al., 1992). High expression of Smad7, as seen in IBD mucosa, was a consequence of deregulated post-translational modification. Various proteins were shown to be involved, including Smurfs, the transcriptional coactivator p300, Arkadia and Jun activation domain-binding protein 1, which regulate Smad7 nuclear export and/or make the protein resistant to proteasome-mediated degradation (Monteleone et al., 2004a; 2004b; 2005). Targeted blocking of Smad7 gene expression was shown to be an effective way to attenuate the ongoing intestinal inflammation (Monteleone et al., 2008). The R-Smad, Smad3 plays an important role in TGFβ signaling. Smad3 heterozygous mice, characterized by reduced levels of Smad3, showed accelerated healing of colonic mucosa after experimental induction of colitis with TNBS. The most prominent effects were on re-epithelization and proliferation of the intestinal epithelium, which was associated with reduced production of TGF-β1 (Tokumasa et al., 2004). Another molecule of this pathway, CTGF, which acts as a downstream effector of the TGFβ signaling pathway, was markedly increased in almost 90% of CD tissue samples. CTGF expression is mostly localized in fibroblasts of the submucosal layer while completely lacking in inflammatory cells (di Mola et al., 2004). More recently, it has been reported that deficiency of Smad5 in intestinal epithelial cells leads to an increase of cell migration and villus lengthening followed by disassembly of the apical junctional complex (Allaire et al., 2011). This intestinal epithelial impairment makes Smad5<sup>Δ</sup>IEC mice more susceptible to DSS colitis development and reduced healing. Decreased expression of the BR-Smad, Smad5 was also found in intestinal samples from IBD patients, which emphasizes the

Several growth factors like TGFβ superfamily, growth hormone, epidermal growth factor, keratinocyte growth factor, teduglutide and granulocyte macrophage/granulocite colony

(Alonzi et al., 2004; Hibi et al., 2002; Mombaerts et al., 1993).

importance of BMP signaling in IBD.

receptor (CaSR), expressed on the epithelia of the GI tract and myofibroblasts, downregulated BMP4 and noggin expression and raises the effective concentration of BMP2 leading to increased intestine repair and barrier development (Peiris et al., 2007).

Fig. 4. BMP signaling components in compartments of the normal colon crypt (from Kosinski et al. (2007). Gene expression patterns of human colon tops and basal crypts and BMP antagonists as intestinal stem cell niche factors. *Proc Natl Acad Sci USA* 104:15418- 15423. Copyright (2007) National Academy of Science, U.S.A. Used with kind permission).

## **3.3 BMPs and TGF**β**/BMP signaling pathway in inflammatory bowel disease**

Animal models of colitis are often used to aid in IBD research. They allow a detailed examination of the pathological process from acute to chronic phase, providing an insight into the immunological disturbances behind the pathogenesis of IBD (Hibi et al., 2008; Mizoguchi & Mizoguchi, 2010; Shi et al., 2011). In these animal models colitis can be induced by chemical, immunological, microbiological and physical factors (Boirivant et al., 1998; Morris et al., 1989; Okayasu et al., 1990). There are also transgenic and knock-out mouse strains that develop colitis, as well as spontaneous colitis models (Matsumoto et al., 1998; Mombaerts et al., 1993; Sadlack et al., 1993; Sundberg et al., 1994). Chemically induced colitis is one of the most utilized animal models. Dextran sulfate sodium (DSS), a heparin-like polysaccharide is often used to induce a UC-like colitis because of its simplicity and reproducibility of the inflammatory intestinal lesions. In the intestine of DSS treated mice epithelial barrier disruption with neutrophil and macrophage infiltration is observed as well as abnormal

receptor (CaSR), expressed on the epithelia of the GI tract and myofibroblasts, downregulated BMP4 and noggin expression and raises the effective concentration of BMP2

leading to increased intestine repair and barrier development (Peiris et al., 2007).

Fig. 4. BMP signaling components in compartments of the normal colon crypt (from Kosinski et al. (2007). Gene expression patterns of human colon tops and basal crypts and BMP antagonists as intestinal stem cell niche factors. *Proc Natl Acad Sci USA* 104:15418- 15423. Copyright (2007) National Academy of Science, U.S.A. Used with kind permission).

**3.3 BMPs and TGF**β**/BMP signaling pathway in inflammatory bowel disease** 

Animal models of colitis are often used to aid in IBD research. They allow a detailed examination of the pathological process from acute to chronic phase, providing an insight into the immunological disturbances behind the pathogenesis of IBD (Hibi et al., 2008; Mizoguchi & Mizoguchi, 2010; Shi et al., 2011). In these animal models colitis can be induced by chemical, immunological, microbiological and physical factors (Boirivant et al., 1998; Morris et al., 1989; Okayasu et al., 1990). There are also transgenic and knock-out mouse strains that develop colitis, as well as spontaneous colitis models (Matsumoto et al., 1998; Mombaerts et al., 1993; Sadlack et al., 1993; Sundberg et al., 1994). Chemically induced colitis is one of the most utilized animal models. Dextran sulfate sodium (DSS), a heparin-like polysaccharide is often used to induce a UC-like colitis because of its simplicity and reproducibility of the inflammatory intestinal lesions. In the intestine of DSS treated mice epithelial barrier disruption with neutrophil and macrophage infiltration is observed as well as abnormal cytokine production (Dieleman et al., 1994; Kitajima et al., 1999). Trinitrobenzene sulfonic acid (TNBS) dissolved in ethanol induces a CD-like colitis, with transmural inflammation (Elson et al., 1995). Ethanol is thought to cause a transient increase in intestinal permeability, allowing TNBS to invade the musocal layers and act as a hapten to form complete antigens with tissue proteins. This provokes an intense and sustained inflammatory response and breaks T-cell tolerance to mucosal antigens (Boismenu & Chen, 2000). Genetic colitis models such as gene knockout and transgene models offer another approach in experimental IBD research. They imply genetic modifications that favor the development of colitis. IL-2 deficient mice spontaneously develop a UC-like colitis in 100% of cases, while mice lacking the IL-10 gene develop chronic colitis only in the presence of enteric bacteria (Kuhn et al., 1993; Sadlack et al., 1993). Other genetic models include T cell receptor, STAT3 and IL-17 gene manipulation (Alonzi et al., 2004; Hibi et al., 2002; Mombaerts et al., 1993).

Monteleone et al. (2001) were among the first groups to show the disturbance of the TGF-β1 signaling pathway in human IBD. They found that Smad7, an inhibitor of BMP-signaling, was overexpressed, whereas Smad3, an R-Smad of this signaling pathway, was downregulated. Due to Smad7 inhibition, lamina propria mononuclear cells (LPMC) isolated from mucosa of CD patients were not able to respond to TGF-β1 and to downregulate proinflammatory cytokine expression, especially of TNFα and IFNγ. Moreover, Smad7 inhibition by specific antisense oligonucleotides allowed LPMC to respond to TGF-β1 restoring TGF-β1 signaling by increasing pSmad3 and decreasing Smad7, and provide TGF-β1-mediated inhibition of proinflammatory cytokine production in experimentally induced colitis (Boirivant et al., 2006). Previously, it was shown that mice with targeted disruption of the TGF-β1 gene or one of its intracellular signaling components develop multifocal inflammation (Shull et al., 1992). High expression of Smad7, as seen in IBD mucosa, was a consequence of deregulated post-translational modification. Various proteins were shown to be involved, including Smurfs, the transcriptional coactivator p300, Arkadia and Jun activation domain-binding protein 1, which regulate Smad7 nuclear export and/or make the protein resistant to proteasome-mediated degradation (Monteleone et al., 2004a; 2004b; 2005). Targeted blocking of Smad7 gene expression was shown to be an effective way to attenuate the ongoing intestinal inflammation (Monteleone et al., 2008).

The R-Smad, Smad3 plays an important role in TGFβ signaling. Smad3 heterozygous mice, characterized by reduced levels of Smad3, showed accelerated healing of colonic mucosa after experimental induction of colitis with TNBS. The most prominent effects were on re-epithelization and proliferation of the intestinal epithelium, which was associated with reduced production of TGF-β1 (Tokumasa et al., 2004). Another molecule of this pathway, CTGF, which acts as a downstream effector of the TGFβ signaling pathway, was markedly increased in almost 90% of CD tissue samples. CTGF expression is mostly localized in fibroblasts of the submucosal layer while completely lacking in inflammatory cells (di Mola et al., 2004). More recently, it has been reported that deficiency of Smad5 in intestinal epithelial cells leads to an increase of cell migration and villus lengthening followed by disassembly of the apical junctional complex (Allaire et al., 2011). This intestinal epithelial impairment makes Smad5<sup>Δ</sup>IEC mice more susceptible to DSS colitis development and reduced healing. Decreased expression of the BR-Smad, Smad5 was also found in intestinal samples from IBD patients, which emphasizes the importance of BMP signaling in IBD.

Several growth factors like TGFβ superfamily, growth hormone, epidermal growth factor, keratinocyte growth factor, teduglutide and granulocyte macrophage/granulocite colony

Bone Morphogenetic Proteins and Signaling Pathway in Inflammatory Bowel Disease 207

(a) Acute stage of colitis (b) Chronic stage of colitis (c) BMP7 treatment

Fig. 7. Representative histological sections of colon from colitic rats treated with vehicle (a, b) and BMP7 (c). Histologic findings in early stage of TNBS-colitis showed intensive inflammatory cell infiltration associated with colonic mucosa swelling and bleeding. In chronic stage of TNBS-colitis (b), diffuse infiltration with lymphocytes and fibroblasts was observed in ulcus region presenting healing process of colon tissue. The microstructure of colon was restored upon therapeutically application of BMP7 (c) Magnification x100.

BMP2 and BMP7 expression has been found in colon samples during acute and chronic stages of experimental colitis. BMP7 treatment slightly reduced the expression level of BMP2 (Maric et al., 2003; 2008). Beneficial effects of BMP7 were also observed in intestinal ischemia/reperfusion injury and fibrosis associated with chronic intestinal inflammation

Intestinal fibrosis is a major complication of CD disease which is an outcome of multiple factors. It occurs as a result of different types of chronic inflammation, initiated in various cellular sources, but all result in deregulation of extracellular matrix turnover (Burke 2007, Fiocchi & Lund, 2011). TGFβ is described as the most potent profibrotic factor of several

 (a) PCNA staining (b) Naphtol AS-D chloroacetate esterase staining Fig. 8. Effect of BMP7 treatment on cell proliferation and neutrophil accumulation in TNBSinduced colitis. BMP7 significantly reduced cell proliferation and neutrophil accumulation

(Flier et al., 2010; Radhakrishnan et al., 2008).

upon 14-day long therapy.

**3.4 BMP and their pathway in intestinal fibrosis** 

stimulating factors have been involved in clinical studies so far (Krishnan et al., 2010). BMPs as osteoinductive agents are mostly implemented in healing bone damage but the presence of their expression during development and in adulthood indicates their importance during different disease and healing processes. We have previously shown the accumulation of radioactively labeled BMP7 in the GI tract primarily in stomach and intestine with the highest uptake in terminal ileum (Fig. 5)(Maric et al., 2003).

Fig. 5. Biodistrubution of 125I-labeled BMP7 in rat gastrointestinal tract. Rats were analyzed at different time points following i.v. injection of 125I-BMP7 (injected dose was 25 μCi/0.791 μg/kg). Relative uptake of 125I-BMP7 was expressed as ng of radiolabeled BMP7/g wet tissue weight (Maric et al., 2003).

In addition, systemically applied BMP7 reduced the macroscopic and microscopic changes observed in TNBS-induced colitis followed by downregulation of proinflammatory cytokines (i.e. IL-6, TNFα, ICAM-1) and pro-fibrogenic cytokines (i.e. TGFβ). BMP7 accelerated healing of wounded tissue and reduced neutrophil infiltration (Fig. 6, Fig. 7 & Fig. 8).

(a) TNBS-colitis (b) TNBS-colitis upon BMP7 treatment

Fig. 6. Macroscopic appearance of colonic mucosa during different days of experimentally induced colitis. The cobblestone-like ulceration in acute (days 2 and 5) and linar ulceration in chronic (days 14 and 30) stage was evident in colon. BMP7, applied systemically reduced macroscopic colon damage.

(a) Acute stage of colitis (b) Chronic stage of colitis (c) BMP7 treatment

stimulating factors have been involved in clinical studies so far (Krishnan et al., 2010). BMPs as osteoinductive agents are mostly implemented in healing bone damage but the presence of their expression during development and in adulthood indicates their importance during different disease and healing processes. We have previously shown the accumulation of radioactively labeled BMP7 in the GI tract primarily in stomach and intestine with the

Fig. 5. Biodistrubution of 125I-labeled BMP7 in rat gastrointestinal tract. Rats were analyzed at different time points following i.v. injection of 125I-BMP7 (injected dose was 25 μCi/0.791 μg/kg). Relative uptake of 125I-BMP7 was expressed as ng of radiolabeled BMP7/g wet

In addition, systemically applied BMP7 reduced the macroscopic and microscopic changes observed in TNBS-induced colitis followed by downregulation of proinflammatory cytokines (i.e. IL-6, TNFα, ICAM-1) and pro-fibrogenic cytokines (i.e. TGFβ). BMP7 accelerated healing

 (a) TNBS-colitis (b) TNBS-colitis upon BMP7 treatment Fig. 6. Macroscopic appearance of colonic mucosa during different days of experimentally induced colitis. The cobblestone-like ulceration in acute (days 2 and 5) and linar ulceration in chronic (days 14 and 30) stage was evident in colon. BMP7, applied systemically reduced

of wounded tissue and reduced neutrophil infiltration (Fig. 6, Fig. 7 & Fig. 8).

highest uptake in terminal ileum (Fig. 5)(Maric et al., 2003).

tissue weight (Maric et al., 2003).

macroscopic colon damage.

Fig. 7. Representative histological sections of colon from colitic rats treated with vehicle (a, b) and BMP7 (c). Histologic findings in early stage of TNBS-colitis showed intensive inflammatory cell infiltration associated with colonic mucosa swelling and bleeding. In chronic stage of TNBS-colitis (b), diffuse infiltration with lymphocytes and fibroblasts was observed in ulcus region presenting healing process of colon tissue. The microstructure of colon was restored upon therapeutically application of BMP7 (c) Magnification x100.

BMP2 and BMP7 expression has been found in colon samples during acute and chronic stages of experimental colitis. BMP7 treatment slightly reduced the expression level of BMP2 (Maric et al., 2003; 2008). Beneficial effects of BMP7 were also observed in intestinal ischemia/reperfusion injury and fibrosis associated with chronic intestinal inflammation (Flier et al., 2010; Radhakrishnan et al., 2008).

## **3.4 BMP and their pathway in intestinal fibrosis**

Intestinal fibrosis is a major complication of CD disease which is an outcome of multiple factors. It occurs as a result of different types of chronic inflammation, initiated in various cellular sources, but all result in deregulation of extracellular matrix turnover (Burke 2007, Fiocchi & Lund, 2011). TGFβ is described as the most potent profibrotic factor of several

(a) PCNA staining (b) Naphtol AS-D chloroacetate esterase staining

Fig. 8. Effect of BMP7 treatment on cell proliferation and neutrophil accumulation in TNBSinduced colitis. BMP7 significantly reduced cell proliferation and neutrophil accumulation upon 14-day long therapy.

Bone Morphogenetic Proteins and Signaling Pathway in Inflammatory Bowel Disease 209

villus architecture and expansion of the stroma with plasma cell infiltrates, while mice with conditional Smad4 deletion in the intestinal epithelial layer did not develop intestinal tumors (Kim et al., 2006). In contrast, polyps from JP patients with a germline SMAD4 mutation showed biallelic inactivation of SMAD4 in both the epithelium and stroma,

Familial adenomatous polyposis (FAP) is an autosomal dominant syndrome characterized by hundreds to thousands of adenomatous colorectal polyps that are caused by a deletion in the adenomatous polyposis coli (APC) gene, localized on chromosome 5q21. Polyps develop in adolescence, and if not treated, malignant alteration inevitably occurs (Morton et al., 1993). Extraintestinal manifestations may also be present in form of osteomas, desmoid tumors, dental abnormalities and extracolonic cancer (Galiatsatos & Foulkes, 2006). APC gene plays an important role in Wnt signaling and is involved in ubiquitine-mediated degradation of βcatenin. Functional defects of APC therefore lead to aberrant Wnt pathway activity, resulting in uncontrolled cell proliferation (Rustgi et al., 2007). In intestinal homeostasis, BMP molecules have a suppresive effect on Wnt-β-catenin pathway and preserved BMP signaling is required for inhibition of intestinal stem cell proliferation and repression of polyp formation (He et al., 2004; Haramis et al., 2004). Conversely, higher expression of Wnt signaling molecules in FAP results in BMP signaling downregulation. In a study on Wnt target genes, in APC mutant mice heterozygous for an allele equivalent to a human APC mutation, more severe polyposis and faster rate of tumor growth was associated with higher expression of the BMP antagonist Gremlin 1 and lower BMP2 and BMP4 expression (Lewis et al., 2010). Analogous results were found in human FAP tissue specimens, with BMP2 expression lost in dysplatic epithelium of

Association of JP and FAP with the BMP signaling pathway was confirmed by experiments on BMP-transgenic mice. Transgenic mice which overexpressed the BMP anatagonist noggin or mice with conditional inacativation of BMPRIA which led to disruption of both epithelial and mesenchymal BMP signaling, showed highly increased formation of intestinal polyps morphologically similar to those in JP (Haramis et al., 2004; He et al., 2004). Loss of mesenchymal BMP signaling by conditional inactivation of BMPRII in stroma led to intestinal bleeding, thickness of mucosa due to epithelial hyperplasia and myofibroblast increment and multiple hemartomatous polyp appearance (Beppu et al., 2008; Hardwick et al., 2008). These expression patterns found in human polyposis syndromes indicate that altered BMP expression plays an important role in uncontrolled cell proliferation and

Finally, alteration of the BMP signaling pathway may be involved in the appearance of intestinal mucosal atrophy due to total parenteral nutrition (Zhang et al., 2009). Expression of BMP2, BMP4, BMPRII and pSmad1/5/8 has been found to be increased in intestinal mucosa without differences in BMP antagonist expression following parenteral nutrition.

Extensive research was conducted to explore the genes and factors that may trigger the initiation and progression of colorectal cancers (Ulman & Itzkowitz, 2011). Among a small number of genes known to affect cancer development, BMPs and their signaling pathway have been under intensive research during the last decade (van den Brink & Offerhaus, 2007). So far, studies have revealed contradictory findings. Some of them have shown reduced or complete

loss of BMP signaling, while others have shown increased expression of this pathway.

These alterations were suggested to affect epithelial cell proliferation.

**3.6 BMPs and their pathway in gastrointestinal cancers** 

suggesting a common clonal origin (Woodford-Richens et al., 2000).

mycroadenomas (Hardwick et al., 2004).

tumorigenesis in the intestine.

organs including organs of the GI tract. Experimental studies revealed that disruption of the TGFβ/Smad signaling pathway contributed to both chronic tissue inflammation and fibrosis (Flanders 2004; Nguyen & Goldschmeding, 2008; Pucilowska et al., 2000; Rieder et al., 2007). Key molecules of this pathway are Smad3 and Smad7. Smad3 null mice exhibit a significant reduction of intestinal fibrosis, associated with increased intestinal expression of Smad7 and decreased expression of Smad3, CTGF, collagen I-III and TGFβ following TNBS induced colitis (Latella et al., 2009; Zanninelli et al., 2006,). Wild-type mice, on the other hand, showed decreased expression of Smad7 and increased expression of Smad3, similar to the findings of di Sabatino (2009) in biopsies of intestinal strictures from patients suffering from Crohn's disease. Myofibroblasts from mucosa underlying CD strictures produced significantly higher amounts of collagen induced by an increased expression of TGFβ and Smad2/3 but not Smad7 (di Sabatino et al., 2009). It was pointed out that a disturbance of TGFβ/BMP downstream signaling components could contribute to intestinal fibrosis and therefore they were presented as more specific antifibrotic targets than TGFβ. Increased expression of TGFβ, CTGF, collagen-1α, and BMP7 was found in strictured segments of CD intestinal biopsies (Burke et al., 2008), although previous studies showed anti-inflammatory and antifibrotic properties of BMP7 (Maric et al., 2003; Flier et al., 2010). The correlation between steroid treatment and stricture development in CD was also observed followed by increased expression of CTGF in both, *in vivo* and *in vitro* by stimulating intestinal fibroblasts (Burke et al., 2008). The capacity of BMP7 to antagonize the pro-fibrotic effects of TGFβ in various organs including the gut has recently been shown (Fiocchi, 1997; Flier et al., 2010; Zeisberg et al., 2005). Treatment with BMP7 inhibits intestinal fibrosis by downregulation of TNF and by inhibition of TGFβ-induced epithelial to mesenchymal transition of the intestinal epithelial cells, which generates activated fibroblasts and contributes to intestinal fibrosis (Flier et al., 2010).

#### **3.5 BMP and their pathway in other gastrointestinal diseases**

Germline mutations in two members of the BMP pathway have been found to cause juvenile polyposis (JP). JP is an autosomal dominant hamartomatous polyposis syndrome. Affected individuals are predisposed to upper gastrointestinal and colorectal cancer. In almost half of the cases, patients have mutations or deletions in *SMAD4* and *BMPR1A* genes (Howe et al., 1998; Houlston et al., 1998; Langeveld et al., 2010; van Hattem et al., 2008; Woodford-Richens et al., 2001; Zhou et al., 2001). Genetic linkage studies of JP patients revealed that Smad4 germline mutation was mapped to chromosome 18q21 while BMPR1A germline mutations were localized on chromosome 10q22-23 (Howe et al., 1998; 2001). In a genotypephenotype correlation study of JP patients, a significant prevalence of gastric polyposis was found in patients with Smad4 mutations when compared to other subsets of patients (Friedel et al., 2002). Histologically, JP polyps are characterized by dilated glands, abundant stroma and inflammatory infiltrates in a thickened lamina propria with normal epithelial covering (Howe et al., 2004). SMAD4 is a known tumor suppressor gene in pancreatic and colon cancer, but in JP it was hypothesized that SMAD4 gene acts as a susceptibility gene, a "gatekeeper", its loss of function resulting in polyp formation through indirect mechanisms, suggesting an important role of the stromal inflammatory response in the regulation of epithelial tumorigenesis (Moskaluk et al., 1997; Takagi et al., 1996). This theory was supported by a study on homozygous Smad4 knockout mice. The most important histological findings included thickened intestinal musoca with polyp formation, loss of

organs including organs of the GI tract. Experimental studies revealed that disruption of the TGFβ/Smad signaling pathway contributed to both chronic tissue inflammation and fibrosis (Flanders 2004; Nguyen & Goldschmeding, 2008; Pucilowska et al., 2000; Rieder et al., 2007). Key molecules of this pathway are Smad3 and Smad7. Smad3 null mice exhibit a significant reduction of intestinal fibrosis, associated with increased intestinal expression of Smad7 and decreased expression of Smad3, CTGF, collagen I-III and TGFβ following TNBS induced colitis (Latella et al., 2009; Zanninelli et al., 2006,). Wild-type mice, on the other hand, showed decreased expression of Smad7 and increased expression of Smad3, similar to the findings of di Sabatino (2009) in biopsies of intestinal strictures from patients suffering from Crohn's disease. Myofibroblasts from mucosa underlying CD strictures produced significantly higher amounts of collagen induced by an increased expression of TGFβ and Smad2/3 but not Smad7 (di Sabatino et al., 2009). It was pointed out that a disturbance of TGFβ/BMP downstream signaling components could contribute to intestinal fibrosis and therefore they were presented as more specific antifibrotic targets than TGFβ. Increased expression of TGFβ, CTGF, collagen-1α, and BMP7 was found in strictured segments of CD intestinal biopsies (Burke et al., 2008), although previous studies showed anti-inflammatory and antifibrotic properties of BMP7 (Maric et al., 2003; Flier et al., 2010). The correlation between steroid treatment and stricture development in CD was also observed followed by increased expression of CTGF in both, *in vivo* and *in vitro* by stimulating intestinal fibroblasts (Burke et al., 2008). The capacity of BMP7 to antagonize the pro-fibrotic effects of TGFβ in various organs including the gut has recently been shown (Fiocchi, 1997; Flier et al., 2010; Zeisberg et al., 2005). Treatment with BMP7 inhibits intestinal fibrosis by downregulation of TNF and by inhibition of TGFβ-induced epithelial to mesenchymal transition of the intestinal epithelial cells, which generates activated fibroblasts and

contributes to intestinal fibrosis (Flier et al., 2010).

**3.5 BMP and their pathway in other gastrointestinal diseases** 

Germline mutations in two members of the BMP pathway have been found to cause juvenile polyposis (JP). JP is an autosomal dominant hamartomatous polyposis syndrome. Affected individuals are predisposed to upper gastrointestinal and colorectal cancer. In almost half of the cases, patients have mutations or deletions in *SMAD4* and *BMPR1A* genes (Howe et al., 1998; Houlston et al., 1998; Langeveld et al., 2010; van Hattem et al., 2008; Woodford-Richens et al., 2001; Zhou et al., 2001). Genetic linkage studies of JP patients revealed that Smad4 germline mutation was mapped to chromosome 18q21 while BMPR1A germline mutations were localized on chromosome 10q22-23 (Howe et al., 1998; 2001). In a genotypephenotype correlation study of JP patients, a significant prevalence of gastric polyposis was found in patients with Smad4 mutations when compared to other subsets of patients (Friedel et al., 2002). Histologically, JP polyps are characterized by dilated glands, abundant stroma and inflammatory infiltrates in a thickened lamina propria with normal epithelial covering (Howe et al., 2004). SMAD4 is a known tumor suppressor gene in pancreatic and colon cancer, but in JP it was hypothesized that SMAD4 gene acts as a susceptibility gene, a "gatekeeper", its loss of function resulting in polyp formation through indirect mechanisms, suggesting an important role of the stromal inflammatory response in the regulation of epithelial tumorigenesis (Moskaluk et al., 1997; Takagi et al., 1996). This theory was supported by a study on homozygous Smad4 knockout mice. The most important histological findings included thickened intestinal musoca with polyp formation, loss of villus architecture and expansion of the stroma with plasma cell infiltrates, while mice with conditional Smad4 deletion in the intestinal epithelial layer did not develop intestinal tumors (Kim et al., 2006). In contrast, polyps from JP patients with a germline SMAD4 mutation showed biallelic inactivation of SMAD4 in both the epithelium and stroma, suggesting a common clonal origin (Woodford-Richens et al., 2000).

Familial adenomatous polyposis (FAP) is an autosomal dominant syndrome characterized by hundreds to thousands of adenomatous colorectal polyps that are caused by a deletion in the adenomatous polyposis coli (APC) gene, localized on chromosome 5q21. Polyps develop in adolescence, and if not treated, malignant alteration inevitably occurs (Morton et al., 1993). Extraintestinal manifestations may also be present in form of osteomas, desmoid tumors, dental abnormalities and extracolonic cancer (Galiatsatos & Foulkes, 2006). APC gene plays an important role in Wnt signaling and is involved in ubiquitine-mediated degradation of βcatenin. Functional defects of APC therefore lead to aberrant Wnt pathway activity, resulting in uncontrolled cell proliferation (Rustgi et al., 2007). In intestinal homeostasis, BMP molecules have a suppresive effect on Wnt-β-catenin pathway and preserved BMP signaling is required for inhibition of intestinal stem cell proliferation and repression of polyp formation (He et al., 2004; Haramis et al., 2004). Conversely, higher expression of Wnt signaling molecules in FAP results in BMP signaling downregulation. In a study on Wnt target genes, in APC mutant mice heterozygous for an allele equivalent to a human APC mutation, more severe polyposis and faster rate of tumor growth was associated with higher expression of the BMP antagonist Gremlin 1 and lower BMP2 and BMP4 expression (Lewis et al., 2010). Analogous results were found in human FAP tissue specimens, with BMP2 expression lost in dysplatic epithelium of mycroadenomas (Hardwick et al., 2004).

Association of JP and FAP with the BMP signaling pathway was confirmed by experiments on BMP-transgenic mice. Transgenic mice which overexpressed the BMP anatagonist noggin or mice with conditional inacativation of BMPRIA which led to disruption of both epithelial and mesenchymal BMP signaling, showed highly increased formation of intestinal polyps morphologically similar to those in JP (Haramis et al., 2004; He et al., 2004). Loss of mesenchymal BMP signaling by conditional inactivation of BMPRII in stroma led to intestinal bleeding, thickness of mucosa due to epithelial hyperplasia and myofibroblast increment and multiple hemartomatous polyp appearance (Beppu et al., 2008; Hardwick et al., 2008). These expression patterns found in human polyposis syndromes indicate that altered BMP expression plays an important role in uncontrolled cell proliferation and tumorigenesis in the intestine.

Finally, alteration of the BMP signaling pathway may be involved in the appearance of intestinal mucosal atrophy due to total parenteral nutrition (Zhang et al., 2009). Expression of BMP2, BMP4, BMPRII and pSmad1/5/8 has been found to be increased in intestinal mucosa without differences in BMP antagonist expression following parenteral nutrition. These alterations were suggested to affect epithelial cell proliferation.

#### **3.6 BMPs and their pathway in gastrointestinal cancers**

Extensive research was conducted to explore the genes and factors that may trigger the initiation and progression of colorectal cancers (Ulman & Itzkowitz, 2011). Among a small number of genes known to affect cancer development, BMPs and their signaling pathway have been under intensive research during the last decade (van den Brink & Offerhaus, 2007). So far, studies have revealed contradictory findings. Some of them have shown reduced or complete loss of BMP signaling, while others have shown increased expression of this pathway.

Bone Morphogenetic Proteins and Signaling Pathway in Inflammatory Bowel Disease 211

Ubiquitous presence of BMPs and their importance in regeneration of many tissues aroused their name from bone morphogenetic protein to body morphogenetic protein (Reddi AH 2005). The BMP pathway showed complexity by numerous ligands and their antagonists, downstream effectors and/or other signaling mediators. In addition, the interaction of the

Animal models of IBD provide a valuable tool in IBD research, and are detrimental for the understanding of the role of BMP signaling in this family of diseases. Since they are readily available and reproducible, they contribute greatly to our knowledge of the complex mechanisms of this chronic and debilitating disease and also facilitate the development of

The BMPs play an important role in a multitude of physiological processes and alterations in their signaling properties have been associated with various diseases. These include the majority of pathologies of the gastrointestinal tract, among which Crohn's disease, ulcerative colitis and various GI cancers. Not surprisingly, BMP signaling has been under extensive investigation as a therapeutic target. We, for example, have previously shown the beneficial effects of BMP7 therapy in an experimental model for colitis, with both anti-inflammatory and antifibrotic effects on colon damage. In addition, possible targets of the BMP signaling pathway for the prevention or treatment of IBD are Smad proteins, including Smad3, Smad5 or Smad7. However, many of the molecular mechanisms behind their functions are yet to be

In summary, the BMP signaling pathway plays a major role in GI diseases and we therefore

Akiyama, Y.; Iwanaga, R.; Ishikawa, T.; Sakamoto, K.; Nishi, N.; Nihei, Z.; Iwama, T.; Saitoh,

Allaire, J.M.; Darsigny, M.; Marcoux, S.S.; Roy, S.A.; Schmouth, J.F.; Umans, L.; Zwijsen, A.;

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growth suppression in colon cancer cells is mediated by p21WAF1 stabilization

BMP pathway with other signaling cascades adds another level of regulation.

revealed and are currently under our and other people's investigation.

present it as a group of molecules of high clinical potential for the future.

microsatellite instability. *Cancer*, Vol.78, No.12, pp. 2478-2484.

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new therapeutic modalities.

**5. References** 

Deregulation of mechanisms controlling expression of Smads was found in human colorectal cancers characterized by selective up-regulation of R-Smads in tumor cells and absence of Smad expression in tumor stroma (Korchynskyi et al., 1999). Co-Smad and I-Smad were expressed in both tumor and normal tissues. BMP2, BMP3 and BMP7 have been found to be growth suppressive for colorectal cancer cells (Beck et al., 2006; Hardwick et al., 2004; Loh et al., 2008). BMP2 exerts growth suppression by increasing p21WAF1 protein levels, inducing p21WAF1 stabilization and not its transcription (Back et al., 2007). BMP/Smad independent pathways, like the RAS/ERK-mediated signaling cascade, interferes with BMP2-induced p21WAF1 stabilization, thus acting as a negative regulator of BMP signaling and arresting growth suppression. The expression of BMPRIA and pSmad1 was found in human colon cancer specimens as well as in several cell lines (Beck et al., 2006). The consequence of the loss of normal feedback inhibition could be seen in the intestinal epithelial cells in JP, where the absence of BMP signaling may lead to enhanced production of BMPs in the lamina propria or in epithelial cells (van den Brink, 2004). In addition, the loss of BMP signaling could also be connected with tumor progression. BMP4 and BMP7 expression has been found to increase with progression of the adenoma-carcinoma sequence and to correlate with a worse prognosis (Deng et al., 2007, Motoyama et al., 2008). The comparison of human adenoma and colorectal cancer specimens revealed the loss of active BMP signaling pathway in 9.1% and 77.7%, respectively, (Kodach et al., 2007; 2008a) which correlated with tumor progression from late adenoma to early carcinoma. The most prominent finding was the loss of BMPRII and Smad4 expression in colorectal carcinoma (Hardwick et al., 2008; Kodach et al., 2007; 2008a) which was confirmed by similar findings in sporadic colorectal carcinoma. The loss of BMPRII expression was strictly correlated with microsatellite instability (Kodach et al., 2008b). On the other hand, it was noticed that BMP signaling was regained in more advanced tumor stages (Kodach et al., 2008a).

Significantly higher BMP7 expression was found in human colorectal cancer than in normal tissues. This expression correlated with parameters of pathological aggressiveness and poorest prognosis (Motoyama et al., 2008). BMP7 showed a divergent role in different pathological changes of colon mucosa (Grijelmo et al., 2007). Whether proinvasive or protective activity will prevail depends on the activity of inflammation, tumor type and grade and the status of the BMP-Smad dependent and independent pathways. BMP7 and its receptors are expressed in normal human colon crypts, aberrant crypt foci in sigmoiditis and in half of colorectal tumors. It is involved in cellular scattering and invasion of premalignant and carcinoma colon epithelial cells probably by BMP-Smad independent pathways. Recent studies have re-emphasized the influence of intact BMP signaling on tumor growth as well as on survival and proliferation of colon carcinoma cells *in vivo* and *in vitro* (Lorente-Trigos et al., 2010). The different levels of BMPs (BMP2, BMP4), Smad proteins (Smad1, Smad4, Smad5, and Smad8) and absence of BMP inhibitor (chordin and gremlin) expression was found in advanced sporadic colon carcinomas. Proliferation and tumor growth of primary colon carcinoma cells *in vivo* can be influenced by altering the activity of BMPRIB. An increase in this activity leads to enhanced tumor growth while its inhibition yields the opposite effect. In this respect BMPs, as members of TGFβ superfamily, resemble the TGFβ signaling pathway in sporadic colorectal cancer (Akiyama et al., 1996; Markowitz et al., 1995).

#### **4. Conclusion**

BMPs play a vital role in embryogenesis and in tissue homeostasis of numerous organs including the GI tract and their malfunction may cause different human disorders. Ubiquitous presence of BMPs and their importance in regeneration of many tissues aroused their name from bone morphogenetic protein to body morphogenetic protein (Reddi AH 2005). The BMP pathway showed complexity by numerous ligands and their antagonists, downstream effectors and/or other signaling mediators. In addition, the interaction of the BMP pathway with other signaling cascades adds another level of regulation.

Animal models of IBD provide a valuable tool in IBD research, and are detrimental for the understanding of the role of BMP signaling in this family of diseases. Since they are readily available and reproducible, they contribute greatly to our knowledge of the complex mechanisms of this chronic and debilitating disease and also facilitate the development of new therapeutic modalities.

The BMPs play an important role in a multitude of physiological processes and alterations in their signaling properties have been associated with various diseases. These include the majority of pathologies of the gastrointestinal tract, among which Crohn's disease, ulcerative colitis and various GI cancers. Not surprisingly, BMP signaling has been under extensive investigation as a therapeutic target. We, for example, have previously shown the beneficial effects of BMP7 therapy in an experimental model for colitis, with both anti-inflammatory and antifibrotic effects on colon damage. In addition, possible targets of the BMP signaling pathway for the prevention or treatment of IBD are Smad proteins, including Smad3, Smad5 or Smad7. However, many of the molecular mechanisms behind their functions are yet to be revealed and are currently under our and other people's investigation.

In summary, the BMP signaling pathway plays a major role in GI diseases and we therefore present it as a group of molecules of high clinical potential for the future.

## **5. References**

210 Inflammatory Bowel Disease – Advances in Pathogenesis and Management

Deregulation of mechanisms controlling expression of Smads was found in human colorectal cancers characterized by selective up-regulation of R-Smads in tumor cells and absence of Smad expression in tumor stroma (Korchynskyi et al., 1999). Co-Smad and I-Smad were expressed in both tumor and normal tissues. BMP2, BMP3 and BMP7 have been found to be growth suppressive for colorectal cancer cells (Beck et al., 2006; Hardwick et al., 2004; Loh et al., 2008). BMP2 exerts growth suppression by increasing p21WAF1 protein levels, inducing p21WAF1 stabilization and not its transcription (Back et al., 2007). BMP/Smad independent pathways, like the RAS/ERK-mediated signaling cascade, interferes with BMP2-induced p21WAF1 stabilization, thus acting as a negative regulator of BMP signaling and arresting growth suppression. The expression of BMPRIA and pSmad1 was found in human colon cancer specimens as well as in several cell lines (Beck et al., 2006). The consequence of the loss of normal feedback inhibition could be seen in the intestinal epithelial cells in JP, where the absence of BMP signaling may lead to enhanced production of BMPs in the lamina propria or in epithelial cells (van den Brink, 2004). In addition, the loss of BMP signaling could also be connected with tumor progression. BMP4 and BMP7 expression has been found to increase with progression of the adenoma-carcinoma sequence and to correlate with a worse prognosis (Deng et al., 2007, Motoyama et al., 2008). The comparison of human adenoma and colorectal cancer specimens revealed the loss of active BMP signaling pathway in 9.1% and 77.7%, respectively, (Kodach et al., 2007; 2008a) which correlated with tumor progression from late adenoma to early carcinoma. The most prominent finding was the loss of BMPRII and Smad4 expression in colorectal carcinoma (Hardwick et al., 2008; Kodach et al., 2007; 2008a) which was confirmed by similar findings in sporadic colorectal carcinoma. The loss of BMPRII expression was strictly correlated with microsatellite instability (Kodach et al., 2008b). On the other hand, it was noticed that BMP

signaling was regained in more advanced tumor stages (Kodach et al., 2008a).

sporadic colorectal cancer (Akiyama et al., 1996; Markowitz et al., 1995).

**4. Conclusion** 

Significantly higher BMP7 expression was found in human colorectal cancer than in normal tissues. This expression correlated with parameters of pathological aggressiveness and poorest prognosis (Motoyama et al., 2008). BMP7 showed a divergent role in different pathological changes of colon mucosa (Grijelmo et al., 2007). Whether proinvasive or protective activity will prevail depends on the activity of inflammation, tumor type and grade and the status of the BMP-Smad dependent and independent pathways. BMP7 and its receptors are expressed in normal human colon crypts, aberrant crypt foci in sigmoiditis and in half of colorectal tumors. It is involved in cellular scattering and invasion of premalignant and carcinoma colon epithelial cells probably by BMP-Smad independent pathways. Recent studies have re-emphasized the influence of intact BMP signaling on tumor growth as well as on survival and proliferation of colon carcinoma cells *in vivo* and *in vitro* (Lorente-Trigos et al., 2010). The different levels of BMPs (BMP2, BMP4), Smad proteins (Smad1, Smad4, Smad5, and Smad8) and absence of BMP inhibitor (chordin and gremlin) expression was found in advanced sporadic colon carcinomas. Proliferation and tumor growth of primary colon carcinoma cells *in vivo* can be influenced by altering the activity of BMPRIB. An increase in this activity leads to enhanced tumor growth while its inhibition yields the opposite effect. In this respect BMPs, as members of TGFβ superfamily, resemble the TGFβ signaling pathway in

BMPs play a vital role in embryogenesis and in tissue homeostasis of numerous organs including the GI tract and their malfunction may cause different human disorders.


Bone Morphogenetic Proteins and Signaling Pathway in Inflammatory Bowel Disease 213

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**12** 

*Poland* 

**Genetic Differentiation of Fungi of the Genus** 

Danuta Trojanowska1, Marianna Tokarczyk1, Małgorzata Zwolińska-

*2Department of Gastroenterology, Hepatology and Infectious Diseases of Jagiellonian* 

Inflammatory bowel diseases (IBD) constitute a group of incurable, inflammatory GI diseases of unknown etiology and the chronic course, with periods of spontaneous disease remissions and aggravations (Muszyński, 2001). This group of diseases includes Crohn`s disease and ulcerative colitis. They are characterized by inflammatory reactions and changes in the structure of intestinal mucosa (Rzeszutko, 2006). IBD etiopathogenesis is not clearly defined. The most essential etiological issues include environmental factors, genetic conditioning, abnormalities of intestinal immunological mechanisms and the bacterial flora (Konturek, 2001). The inflammation which develops in the intestine is mostly due to improper, low-fiber diet which is based on highly processed products and on the poor physical activity. Familial diseases confirmed in 10% of cases speak for the genetic source. Immunological mechanisms which develop in response to food and bacterial antigens are extremely significant (Muszyński, 2001; Polińska et al., 2009). A markedly increased interest has been observed in the intestinal microflora which is believed to be essential in inducing IBD development and in relapses of its clinical symptoms. Bacteria which are normally resident in the GI tract determine the proper work of intestines and protect from excessive proliferation of undesired microorganisms. The contribution of the intestinal flora to the development of the biofilm, which prevents the colonization of pathogens including fungi of the genus *Candida* in the GI tract, is extremely significant. The area of interest in fungi which are a part of microflora and constitute a potential source of systemic dissemination by colonizing the GI tract has greatly increased lately (Berhardt & Knoke, 1997). In the normal environment fungi are in balance with the bacterial flora and as commensals do not induce the inflammation. The main cause of its development, however, is the immune system impairment and a decrease of immunity. It affects patients with neoplasms and transplant recipients (Pfaller & Diekema, 2007; Warnock & Campbell, 1996). Other diseases causing inflammations are: diabetes and other endocrinopathies, infections due to HIV virus and also surgical procedures, damaged tissues and inflammations of the

**1. Introduction** 

Wcisło2, Paweł Nowak1, Sebastian Różycki1 and Alicja Budak1

*Candida* **Isolated from Patients with** 

**Inflammatory Bowel Diseases** 

*1Department of Pharmaceutical Microbiology of Jagiellonian* 

*University Collegium Medicum* 

*University Collegium Medicum* 

