How adenomyosis causes infertility?

Tasuku Harada Professor and Chairman

Department of Obstetrics and Gynecology Tottori University Faculty of Medicine　

! Association of Adenomyosis and Infertility

! Inplantatin Factors in Adenomyosis

How Adenomyosis Causes Infertility? �

Prevalence of Patients with Adenomyosis, Endometriosis and Fibroma in Japan (JSOG 2014)

Adenomyosis Endometriosis Myoma

Estimated number of patients who received treatment

131,946 222,218 552,441

Number per 1000 women

4.2 7.1 17.6

Age (y) 43 ± 6.3 38.4 ± 8.0 44.6 ± 8.5

Menarche (y) 12.3 12.2 12.3 Dysmenorrhea % 62.9 56.2 25.2

Dyspareunia % 19.4 18.6 5.7

Infertility % 16.3 14.0 13.0

Age of diagnosis (y) 38.2 34.4 40.6

*

The number was estimated from the survey of patients who visited hospitals and clinics in Japan during 10 days of June 2014. Number of female population aged 20-59 yrs in 2013 was 31,322,000.

*

Estimated number of endometriosis patients who received treatment in Japan

1997 vs 2014

2014 1997

Adenomyosis and associated disease

Prevalence Patients with Adenomyosis, Endometriosis and Fibroma in Japan (JSOG 2014)

Age distribution /100,000

+ Myoma Adenomyosis

+ Myo & Endo + Endometriosis

Clinical evidence of positive correlation between adenomyosis and infertility

1)   Association of dysfunctional uterine JZ and reduced fertility outcome

2)   Achieving pregnancy after being treated with GnRH agonist /Surgery

3)   Negative effects of adenomyosis on reproductive outcome after endometriosis surgery

4)   Compromised ART (IVF/ICSI) outcome in patients with adenomyosis

5)   Association with early miscarriage

6)   Lifelong primary infertility in baboons �

Chiang et al. (1999), Piver et al. (2005), Maubon et al. (2010), Tremellen et al. (2011) �

Medical-Hirata et al. (1993), Igarashi et al.(2000), Nelson and Corson et al. (1993), Silva et al. (1994), Huang et al. (1999), Lin et al. (2000), Wang et al. (2009), Al Jama et al. (2011)

Ballester et al.( 2012)

Tomassetti et al.( 2013), Vercellini et al. (2014), Salim et al. (2012), Maubon et al. (2010), Tremellen et al. (2011), Thalluri et al.( 2012), Tavmergen et al. (2007)

Salim et al. (2012), Martı´nez-Conejero et al. (2011) �

Barrier et al. (2004) �

Percentage (%)

*

*

*

*

Ongoing Pregnancy

Reproductive outcomes in women with adenomyosis after IVF

Conrols Adenomyosis

Endometriosis only A + Endometriosis

Proposed mechanism of infertility in patients with adenomyosis

1. Abnormal utero-tubal transport1) Intra-uterine abnormalities �2) Uterine peristalsis �3) Destruction of normal myometrial architecture and function

2. Altered endometrial function and receptivity �1) Aberrant intra-endometrial metabolism �

i) Altered intra-endometrial steroid metabolism � ii) Abnormal inflammatory response� iii) Altered expression of estrogen and progestrone receptors �

2) Altered uterine oxidative stress environment �3) Impaired implantation �

i) Lack of expression of adhesion molecules � ii) Reduced expression of implantation markers � iii) Altered function of genes for embryonic development �

Publications Factors affected Effect

Ota et al. (1998) Ota et al. (1999) Ota et al. (2001) Ota et al. (2002) Yang et al. (2005) Ulukus et al. (2006) Li et al. (2006) Yen et al. (2006) Lessey et al. (2006)

Nitric oxide synthase (NOS) Superoxide dismutase (SOD) Xanthine Oxidase Catalase Interleukin-6 Interleukin 8 receptors CXCR1 and CXCR2 Matrix metalloproteinases ( MMP2 and MMP9), Vescular endothelial growth factor (VEGF) Microvessel density (MVD) Leukaemia inhibitory factor (LIF) LIF-receptor α Interleukin-11 Cytochrome P450

Increased Increased Increased Increased Increased Increased Increased Increased Decreased Decreased Decreased Increased

Endothelium of endometrium Glandular and surface epithelium of endometrium Both eutopic and ectopic endometrium Both eutopic and ectopic endometrium Macrophage co-cultured ESCs Eutopic endometrium Both eutopic and ectopic endometrium Ectopic endometrium Both eutopic and ectopic endometrium Both eutopic and ectopic endometrium Both eutopic and ectopic endometrium Eutopic endometrium

Various factors with altered expression in adenomyosis

Publications Factors affected Effect

Wang et al. (2009) [3] Wichereck et al. (2009) Tokyol et al. (2009) Goteri et al. (2009) Yang et al. (2009) Xiao et al. (2009) Xiao et al. (2010) Fischer et al. (2011) Qin et al. (2011) Xiao et al. (2013)

Interleukin-10 RCAS1 Matrix metalloproteinase-2 Hypoxia-inducible factor-1 α (HIF-1 α ) Vescular endothelial growth factor (VEGF) Matrix metalloproteinase-2 (MMP-2) ,Tissue inhibitor of metalloproteinase – 1 (TIMP-1) Integrin β 3 and Osteopontin (OPN) Leukemia inhibitory factor (LIF) HOXA 10 Interleukin-10 R1 and Interleukin-10 R2 Integrin β 3 and Osteopontin (OPN)

Increased Decreased Increased Increased Increased Increased Decreased Decreased Decreased Increased Decreased

Both eutopic and ectopic endometrium (epithelial cells) Serum concentration level Ectopic endometrium Ectopic endometrium Both eutopic and ectopic endometrium Endometrial stromal cells Eutopic endometrium Uterine flushing fluid and endometrium Eutopic endometrium Eutopic endometrium Eutopic endometrium

Table. Continued

Eutopic endometrium

LIF IL-11 HOXA-10 IGβ3 OPN

NO SOD XO Catalase VEGF MMP2 TIMP-1 IL-10 IL-8R P450

Biological factors showing altered expression in adenomyosis

Adenomyotic lesion

XO Catalase VEGF MMP2 IL-10 IL-6 HIF-1 α MVD

LIF IL-11

LIF was the first cytokine shown to be critical for implantation in mice.

! Association of Adenomyosis and Infertility

! Implantation Factors in Adenomyosis

How Adenomyosis Causes Infertility? �

IL-6, IL-8, TNFa RANTES, LIF, GROa, OPN, MCP1

Implantation and Inflammatory Reaction

During implantation, expression of various kinds of hormones, cytokines, growth factors, and matrixsubstances has been reported in the uterus. These molecules are under strict temporal and spatial regulation of maternal hormones and probably fetus-derived substances.Implantation is also characterized by activation of pro-inflammatory NFkB pathway leading to an increase in the levels of cytokines, PGs and MMPs.

Modified from Dekel N et al. 2010

Staun-Ram et al. 2005

During invasion, trophoblast cells break through the epithelial and stromal cells. �The endometrial tissue is then repaired and remodeled by the growing placenta. �This local ‘wound healing-like’ process is characterized by a strong Th1, pro-inflammatory response in which high levels of cytokines such as IL6, LIF, IL8, and TNFa are involved.�

Gene � Female reproductive phenotype� References �CSF-1� Increased fetal resorption � Pollard et al. (1991) �

GM-CSF � Mean litter sizes small, placenta deficiency � Robertson et al. (1999) �

IL-1Rt1 � Implantation normal, minor effect on litter size�

Abbondanzo et al. (1996) �

IL-6 � Reduced fertility, viable implantation rates decreased 48% �

Robertson et al. (2000) �

IL-11R � Failure of implantation, defective decidualization �

Bilinski et al. (1998) �

gp-130 � Intrauterine lethality, placental deficiency � Yoshida et al. (1996) �

LIF � Failure of implantation � Stewart et al. (1992) �

LIFR� Intrauterine lethality � Ware et al. (1995) �

SOCS3� Placenta defects, embyonic lethality � Roberts et al. (2001) �

Cytokine or cytokine-receptor knockout studies: effects on implantation

Dimitriadis et al. Hum Reprod Update 2005

LIF was the first cytokine shown to be critical for implantation in miceIL-6, IL-11 and LIF are gp130 cytokines which share an accessary signal transducing subunit. �

IL-6 may have an important role in human implantation.

IL-6

IL-1a

#�#�

#�#�

# p<0.05 vs control�

Control

Adenomyosis

#�

Canonical NFkB signaling pathway

Kumar A et al. 2004

NKkB is a transcription factor and critical regulator of innate immunoresponse and inflammation.NF kB is present in its inactive state in the cytoplasm by binding with inhibitory kappa B (IkB).When it is activated by various factors (phosphorylated), IkBα is degraded and p50-p65 heterodimer is translocated to nucleus, then binds DNA and activates the target genes (>150).

IL-6 and LIF expression was increased in endometrial cells via NF-kB activation.

Gene expression IL-6 LIF

Five different members identified in mammals forming homo or heterodimer

p65

p50

NFkB is mainly p65 and p50 heterodimer.

Immunohistochemical staining of�NFkB and phospholylated NFkB in the eutopic endometrium of secretory phase

NFkB (p65)�

Phospho-NFkB (p65)�

Antibodies for p65and phoshpolylated-p65 clearly showed activation of NFkB in the endometrium.

Eutopic Endometrium (Secretory phase)

Adenomyosis -Em

Normal-Em

(X100)

NFkB(p65) � Phospho-NFkB(p65) �

Endometriosis

-Em

NFkB (p65) expression in Secretory Phase Endometrium

(X400) (X100) (X400)

Adenomyosis -Em

Normal-Em

Phospho-p105�

Endometriosis

-Em

NFkB (p105) expression in Secretory Endometrium

(X100) (X400)

Adenomyosis -Em

Normal-Em

(X100)

IL-6 � LIF�

Endometriosis

-Em

IL-6 and LIF expression in Endometrium

(X400) (X100) (X400)

NFkB DNA binding, phosphorylated NFkB and IL-6 expression were down-regulated in eutopic endometrium of endometriosis women. �NFkB DNA Binding

Phosphorylated NFkB

IL-6 mRNA and protein

Transient transfection of IkB alpha mutant (IkBa M) cDNA inhibited NFkB activity in the uterus.

Number of implantation sites was decreased in IkBa M treated mice, resulting in delayed implantation.

Control IkBa M

Co-transfection of LIF cDNA with IkBaM in the uterus partially rescued the delay of implantation induced by suppression of NF-kB activity.

Conclusions

!  Infertility is frequently seen in patients with adenomyosis.

!  Accumulated clinical evidence suggest a positive correlation with

adenomyosis and infertility.

!  Based on published reports, abnormal utero-tubal transport and

altered uterine receptivity may be linked to infertility.

!  Reduced expression of IL-6 and LIF in eutopic endometrium due to decreased activation of NFkB may be the mechanistic cause of infertility in women with adenomyosis.