Molecular Mechanism of Cervical Ripening

WELCOME TO MINOR CREDIT SEMINAR

Molecular Mechanism Of Cervical Ripening in Ruminants

Dr Rouf RashidROLL NO: M-5606

MVSC scholar Animal Reproducion

division

Introduction Stages of cervical remodelling Factors involved in cervical remodelling Role of MMPs Conclusion

Overview

predominantly of

connective tissue

small amounts of smooth muscle tissue presents

cervix is a fibrous organ

thick wall and

constricted lumen

(Leppert 1995).

Introduction

Cervix also having same 3 layers stromal layer more predominant

Mucosal layer:

consisting of

epithelium and

underlying stroma

Serosal layer:

outer most

Muscular layer:

consist of inner

circular & outer

longitudinal

HISTOLOGY OF FEMALE GENITAL TRACT

Cross sectional view of the bovinecervix

3: Cervical lumen with protruding annular rings

4: Epithelial and superficial stromal layer

5: Deep stromal layer 6:Outer muscular layer 7: Serosa

Connective tissue

Ground substance Proteoglycan• Hyalurunic acid,• Chondroitin-4,6

sulfate,• Dermatan sulfate,• Heparan sulfate,&• Keratan sulfate Glycosaminoglycans Fibrillar collagen Matricellular

proteins

Fibrous constituents-

Collagen

Elastin

Reticulin

Cellular elements-

Mast cells

Fibroblasts

Wandering cells

Cervical stroma

I. Gradual ripening- last months ( cervical softening )

II. Final ripening- parturition ( cervical ripening and cervical dilatation)

Gradual cervical ripening-

Clinically - palpable softening

Biochemically – water imbibition, separation of collagen bundles collagen denaturation

Collagen denaturation - cervical MMPs (van Engelen2008)

Cervical Remodelling- 2 stages

Final cervical ripening

Just before and during cervical dilatation.

Collagen fibrils are denatured and digested by matrix metallo proteinases as a part of inflammatory cascade

Cervical softening

Cervical ripening

First measurable increase in tissue distensibility when compared with non pregnant cervix ( Read et al . 2007 )

High P4/E2 ratio

Changes in the ratio of type 1 to type 3 collagen , alteration in post transcriptional processing of collagen monomers and assembly of collagen monomers into collagen fibrils can affect collagen structure and its mechanical strength (Lieu et al. 1997 )

Increase in collagen solubility with no change in total collagen content

Cervical softening

Evidence of downregulation of specific steps in the collagen processing pathway icclude folowing

(a) Decline in cervical expression and activity of lysyl hydroxylase(LH) and lysyl oxidase (LOX) respectively in early pregnancy ( Akins et al . 2011 )

(b) subsequent decline in collagen containing the stronger pyridinoline cross links in second half of gestation

(c) Collagen turnover appears upregulated during the pregnancy but the total collagen content remains constant throughout pregnancy

(d) MMP activity not reported in this phase

(e) Constant breakdown of collagen to maintain steady collagen levels and facilitate replacement of mature by poorly cross linked collagen fibre

Matricellular Proteins

Thrombospondin 2 (thbs2) and tenascin 2 (Tns) proteins are down regulated in early gestation and remain low untill just before birth ( Akins et al . 2011 )

They have a role in cervical softening , through the formation of appropriately assembled collagen fibril and regulate the activity of MMPs , and is a inhibitor of angiogenesis in various tissues ( Kokenyesi et al . 2004 )

Tenascin is upregulated during tissue injury ( postpartum) through proinflammatory and fibrotic responses

Thus replacement of mature cross linked collagen with collagen harbouring reduced cross links with decline in the THBS2 and TNC in the cervix is a key in modulating collagen architecture within ECM during softening

Cervical epithelial

provide immunoprotection and maintenance of a barrier which protect stromal compartment

Trefoil factor 1 (TFF1) is a gastric secreted peptide , protection and restitution of cervical mocosal epithelia ( Read et al . 2007 )

Serine protease inhibitor Kazal type 5 (SPINK5) functions to prevent degredation of desmoglein involved in barrier formation

They are of short duration and very rapid , overlapping each other

Low P4/E2 ratio present

Maximum loss of tissue compliance and structural integrity

Decline in collagen content ( Rajabi et al. 1991 )

Cervical ripening and dilatation

Progressive increase in collagen fibril diametre in the cervix from early to late gestation along with increased spacing between the fibrils ( Akins et al.2011 )

Increased synthesis of glycosaminoglycan and hyaluronan ( HA; Straach et al .2005 )

Proliferation of mucosal epithelia which is dependent on actions of Relaxin and Estrogen ( Lee et al . 2005 )

Epithelium become laden with mucus secreting vacuoles important in immune surveillance and lubrication

Epithelium also expresses steroid 5 α reductase type 1 (SRD5A1) important in local progesterone metabolism ( Mahendroo et al . 1999 )

Loss of p4 function along with increased metabolosim facilitate transition to ripening phase

During ripening the barrier properties of mucosal epithelia , hydration , cell to cell communications are regulated by increased expresion of tight junction proteins (claudin 1 and 2) , aquaporins , gap junction proteins ( connexin 26 and 43 ) , and desmogleins ( Gonzalez et al . 2009 )

Immune cells play a role in ECM remodelling via activation of proinflammatory signals and the release of MMPs that breakdown collagen ( Liggins 2006 )

During ripening there is an influx of monocytes into the cervix

Physiological ripening is not mediated by an inflammatory cascade

Recent studies suggest that eosinophils promote the differentiation of the monocytes into alternatively activated macrophages (M2) during postpartum repair

Expression of epithelial specific genes such as Desmogleins and Keratin 16 as well as inflamatory associated genes and genes involved in ECM synthesis , processing and repair

In contrast to ripening phase , myeloid derived immune cells play an important role in this phase of remodelling

Postpartum cervical repair

Monocytes entered the cervix during ripening phase are differentiated to macrophages and polarised to both M1 and M2 phenotypes with onset of labour or postpartum

Matrix debris clear up

Increased activity of HA metabolising enzyme , hyaluronidase 1, and subsequent increase in low MW HA (Akgul et al .2012 )

Cervical ripening

Enzymatic degradation

Hormonal regulation

Increase in

proteoglycan

Inflammatory mechanism

↓P4

↑E2

↑RELAXIN

PROSTAGLANDIN

Hormonal regulation

Progesterone:

P4 or con. of P4 receptors cervical ripening

Activation of NFκB in turn

Inhibit P4 receptor activity TIMP’S

cervical ripening

.

Relaxin Relaxin stimulates MMPs expression on cervix. promotes both epithelial cells and stromal cells

accumulation

By stimulating cell proliferation& inhibiting apoptosis

Relaxin act on fibroblastic cell receptors

Relese of MMPs causes collagenolysis( TIMP’S)

Mechanism of relaxin induced hydration in cervix not understood.

But, Enlargement of cervial arteries my be a factor and it stimulate procollagenase and also reduces TIMP’s

Relaxin may act cervix through stimulation of PG synthesis

Prostaglandin

Prostaglandin have important role in cervical ripening PGE2 → Chemokine (IL8) → Neutrophils → degranulation → MMPs →

collagen degradation

A.-R. Fuchs et al. / Prostaglandins & other Lipid Mediators 70 (2002) 119–129

Estrogen Estrogen receptors are expressed in cervix at term.

sensory neurons have also been implicated in cervical ripening.

substance P release from neurons terminating in the cervix and spinal cord peaks before parturition.

mRNA levels of calcitonin gene related peptide,which colocalizes with substance P increases at term and its expression increased by estrogen

Trends in Endocrinology and Metabolism 21 (2010) 353–361

Withdrawal of P4 + Proinflam stimulus Act NFkB (Schmitz et al. 2004)

GAGCYTOKINES

-CERVIX

MACROPHAGE-FETAL

iNOs

COX2

NOPG

IL 8neutrophi

ls

IL 1,TNFα IL 8

FIBROBLAST

MMPsNEUTROPHIL

Inflammatory mechanism

Matrix Metalloproteinase (MMPs) play important role in cervical ripening

MMPs secreted by 1.Stromal cells

2.Fibroblasts

3.Smooth muscle cells

4.Inflammatory cells invading cervix(final)

Enzymatic degradation

Cervical total GAG content with progression of pregnancy &

accompanied by dramatic changes in composition.

Function of proteoglycans : 1.Decorin modulate the collagen fibril & spacing

and access to proteases.2. Versican It influences structural disorganization

of the ECM

Hyaluronan synthase 2 expression & subsequent in Hyaluronic acid (HA) is a distinct feature of cervical ripening.

Proteoglycans

(Mahendroo,et al 1996) Trends in Endocrinology and Metabolism 21 (2010) 353–361

In the first step (during ripening), hyaluronic acid & its association with proteoglycan ( versican) is required

Just prior to or during the onset of labor The expression of Hyaluronidase & ADAMTS1 leads

to the disruption of HA–versican cross links going to breakdown in to HA and versican, respectively

The breakdown to smaller size products might be a second step in the loss of cervical tensile strength and integrity required for cervical dilation and birth

(Mahendroo,et al 1996) Trends in Endocrinology and Metabolism 21 (2010) 353–361

(Mahendroo,et al 1996) Trends in Endocrinology and Metabolism 21 (2010) 353–361

(Mahendroo et al. 1999). Trends in Endocrinology and Metabolism 21 (2010) 353–361

conclusion

Incremental and progressisive changes in the ECM occur in a P4 rich environment during softening with further rapid and more aggressive changes to the ECM upon the loss of P4 function during cervical ripening and dilatation

To complete loop of remodelling and allow subsequent pregnancies, proinflammatory and immunosupressive processes modulate tissue repair postpartum

Dynamic, cumulative changes in the crvical ECM are initiated early in the pregnancy and culminate in the formation of highly disorganised ECM of unique composition that allows for the loss of tissue integrity resulting in cervical opening during parturation