GOOD MORNING

The Endometrial cycle Prepared by Dr. RahulDepartment of PhysiologySMS Medical College Jaipur

OUTLINES OF TOPICS Histology and functional component of the

endometrium- why we to learn histology inPhysiology?

SONOGRAPHIC FINDINGS in different phases of the normal endometrium cycle.

In-vitro fertilization

Role of ENDOMETRIUM related Infertility and how to come up with this limitation.

Importance of endometrium in treatment of DUB

A CASE STUDY : A novel approach to treat endometrial related infertility in near future.

What is Menstruation?

Menstruation is the failure of reproduction ;Picturesquely said that

the “uterus weeps a bloody tear” when a fertilized ovum does not

materialize.

Sequence of events in the ovarian and menstrual cycles may

be briefly summarized:Ovary Graffian Follicle Ovulation corpus luteum

Oestrogens Progesterone

Endometrium Proliferation Secretion Menstruation

This cycle continues throughout reproductive life unless interrupted by childbirth or disease.

Source : Reproductive Physiology For Medical Students : Philip Rhodes :J & A Churchill Ltd 1969 :Page 55.

Endometrium is mucosal lining of the uterine cavity (Williams Obstetrics 21st ed/66)

It is the anatomical site of blastocyst apposition ,

implantation and fetal/placental development but cannot

claimed as unique property as of ectopic pregnancies.

(Williams Obstetrics 21st ed/66)

Endometrium is one principal target tissue of the

pituitary-gonadal axis, but has also been recognized as

an endocrine organ itself. (journal.1.)

Endometrium is formed by fusion of the mullerian ducts

between 8th and 9th post ovulatory weeks. (Blaustein's

Pathology/5th ed /chap9/383)

Until 20th week of gestation the endometrium is composed of

single layer of columnar epithelium supported by a thick layer of

fibroblastic stroma. (Blaustein's Pathology/5th ed /chap9/383)

By 20th week of gestation ,the surface epithelium invaginates

into the underlying stroma forming glandular structures that

extend towards the underlying myometrium.(Blaustein'sPathology)

EMBRYOLOGY OF ENDOMETRIUM

At birth uterus measures about 4 cm in length much of

which is made of cervix .the endometrial surface and

glands are lined by low columnar to cuboidal

epithelium ,which resembles as inactive endometrium

seen in menopause. (Blaustein's Pathology/5th ed /chap9/383)

The endometrium during the reproductive period

undergoes cyclic morphologic changes. (Blaustein's

Pathology/5th ed /chap9/383)

THE HISTOLOGICAL APPEARANCE OF ENDOMETRIUM

2/3rd

1/3rd

Histologically it is divided into two layers (Dutta’s textbook gynaecology;chap7/82)

Stratum basalis :

1/3 rd of total depth of the endometrium and lies in contact with the

myometrium.

Uninfluenced by hormones and no cyclic changes occur .

Supplied by basal arteries .

After shedding of superficial part during menstruation the regeneration

occurs from this zone.

It measures about 1mm.

Stratum functionalis :

Superficial 2/3rd of the endometrium that proliferates and ultimately

shed if pregnancy does not occur.

This zone is under the influence of fluctuating cyclic ovarian

hormones ,estrogen and progesterone.

The Uterine Endometrial Cycle can be divided into four phases: (Dutta’s textbook of gynaecology;chap7/83)

Stage of Regeneration

Proliferative (Preovulatory or follicular or estrogen )phase

Secretory (Postovulatory or luteal or progesterone ) phase

Mestrual phase

By convention first day of vaginal bleeding is called Day 1 of the

menstrual cycle.(Novak’s Gynecology 13th ed/159)

The first 4 days are occupied with menstruation.The remaining 24

days of the endometrial cycle consists of Proliferative and Secretory

phase. (Jeffcoate’s Gynecology 7th ed/79)

Proliferation Phase:

Extends from 5th or 6th day to 14th day .(1)

Proliferative changes occurs due to rise in level of

ovarian estrogens.(1)

Proliferation of all elements at first slowly but later on

rapidly.(2)

In early proliferative phase (Day 5 to 7) :

the endometrium is less than 2mm in thickness with

narrow straight glands.(2)

Glandular epithelium is composed of cubo-columnar

cells with moderately basophilic cytoplasm and oval or

rounded nuclei in which chromatin is coarse.(8)

Occasional mitoses in both glandular epithelium and

stroma.(5&8)

Stromal cells are oval to elongated with scanty

cytoplasm and oval ,rounded nuclei in which mitoses

are seen.(8)

In mid proliferative and late proliferative phase :

Proliferative activity reaches its maximum between 8th -10th

days of the cycle and by then glandular epithelium is taller.(8)

Epithelium becomes columnar and frequent mitosis is seen

in both glands and stroma .(1,8)

The glandular epithelial cells increases in size and become

pseudostratified(4)

Pseudostratified results because the resting nuclei

occupy basal position while actively dividing nuclei

occupy apical position(4)

Late proliferative phase(Day 11-14 of cycle )

:

The glandular growth out-strips that of the stromal as a

result glands become more convoluted and tortuous. (8)

Glandular epithelium shows marked Pseudostratification(4)

There is increase in stromal ground substance ,which is

edema . (3)

Source :-Internet:http://faculty.une.edu Source:-Williams Obstetrics : 23rd ed/fig.3.2A

Longitudinal view showing coiling of glands Source:Internet:http://Surgpath 4u.com

Pseudostratification :Source :Endometrial cytology with tissue correlations :Maksem .J.A et al:2009: Springer

Under the influence of Estrogen in the proliferative phase of

the cycle , the predominant activity is that of growth reflected

by increase in RNA in the glandular epithelium . (8)

The evidence of cellular proliferation has been demonstrated

by presence of Ki-67. (8)

Estrogens induces synthesis of growth factors like insulin-

like growth factors (IGFs, also called somatomedins;), TGFs,

and epidermal growth factor (EGF). These autocrine and

paracrine mediators are necessary for maturation and growth

of the endometrium. (6)

Tissue breakdown and Apoptosis is not characterstic in

Proliferative phase. (8)

Factors such as bcl-2 which blocks the apoptosis

pathway and causes cell persistence is high while factors

which promotes apoptosis like M30 reactivity are low but

in contrast ,in Secretory Phase the bcl-2 is low while M30

reactivity is high. (8)

Increased mitotic activity of the stromal and glandular

epithelium continues throughout the follicular phase of the

cycle and beyond, until approximately 3 days after ovulation(6)

The thickness of the endometrium increases from about 0.5 to

as much as 5 mm during the proliferative phase.(6)

Levels of estrogen rise early in the follicular phase and peak just

before ovulation.(6)

Levels of endometrial estrogen receptor are highest during the

proliferative phase and decline after ovulation in response to

changing levels of progesterone. (6)

Estrogen induces synthesis of progestin receptors in

endometrial tissue. (6)

Levels of progestin receptors peak at ovulation, when

estrogen levels are highest, to prepare the cells for the

high progestin levels of the luteal phase of the cycle. (6)

Estrogen causes the stromal components of the

endometrium to become highly developed. (6)

Progesterone, opposes the action of estrogen on the

epithelial cells of the endometrium and inhibits epithelial

cell proliferation. (6)

Day to day dating of the endometrium by histological

criteria is not possible during proliferative phase because of

considerable variation among women in the length of pre

ovulatory phase of the cycle .Secretory phase of cycle is

constant in duration.(3) is dictated by the fixed lifespan of the

corpus luteum. (7)

In normal fertile women the follicular phase may be as

short as 5 to 7 days and as long as 21 to 30 days. (3)

Adapted from:-Di Fiore’s Atlas of Histology with Functional Correlations: Victor P. Eroschenko : Lippincott Williams & Wikkins : 12th Edition ;Chapter 21:Page 525-531

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Secretory Phase:

Secretory phase of endometrial cycle starts at

ovulation.(4)

The changes in the components are due to combined

effects of estrogen and progesterone liberated from the

corpus luteum after ovulation .(1)

The Progesterone can only act on the endometrium

previously primed by estrogen (1 & 3)

During this period the endometrium continues to grow

and reach a thickness of around 7 mm .Postovulatory

first 3 days the epithelial cells still undergo mitosis

thereafter mitosis ceases in the glandular epithelium but

can be seen in the stromal cells. (2)

By the Day 17 of the cycle , glycogen accumulates in

the basal portion of glandular epithelium creating

subnuclear vacuoles . This is the first sign of

ovulation that is histologically evident likely due to

direct progesterone action through receptors expressed

in glandular cells. (3)

Day 18, vacuoles move towards the apical portion of

the glandular lumen and pushes the nucleus to the base

of the cell. (3 & 2)

By Day 19-22 , the secretion enters the gland lumen .

This secretion is rich in glycogen ,fructose ,glucose and

has nutritive function for any fertilized ovum reaching the

uterus . (2)

Glandular cell mitosis ceases with secretory activity on

Day 19 due to rising progesterone levels, which

antagonize the mitotic effects of estrogen. (3)

Progesterone exerts its primary antiestrogen effects by

stimulating 17β-HSD and sulfotransferase, enzymes that

convert estradiol to weaker compounds which are

biologically less active compounds. (Journal.2& 3& 6)

Postovulatory day 6-7 (Day 20-21 by cycle )

secretory activity of the gland is maximal and the

endometrium is optimally prepared for

implantation of the blastocyst. (9) . This period has

been called The Implantation Window (Journal.2 & 3)

Subnuclear vacuoles(Pianokey appearance)

Subnuclear vacuoles

B. Early secretory phase

Intraluminal secretions

C. Late secretory phase

Ultrastructurally, pinopodes are evidenced during this

period, indicating that the endometrial surface epithelium is

receptive to the eventual implantation of a blastocyst.

(Journal.2 & 3)

The endometrial glands are engorged with

secretions,so they are no longer straight; instead, they

become tortuous and also because of increased pressure

from surrounding stroma the glands wall infold so they

appear corkscrew and finally saw toothed in

longitudinal section. (6,8,2)

This period is also associated with the secretion of a

certain number of progesterone regulated proteins. (Journal.2.)

Immunohistochemical studies have located these proteins

exclusively in the epithelial component. (Journal.2.)

They are: progesterone-associated endometrial protein

(PEP) , insulin growth factor binding protein 2 (IGFBP2) or

protein 14 or glycodelin ,crystalloglobulin , the integrins

and glycoproteins or type 1 mucins which are secreted into

the cytoplasm of the glands. (Journal.2.)

The physiological role played by these various proteins

is still not fully understood. (Journal.2.)

PP14 would seem to have role in facilitating embryo

implantation.

Evidence:-The uterine flushings of patients who

have had miscarriage show reduced PP 14

secretion. (Journal.2.)

Mucin, particularly muc-1, has important functions at

the luminal surface and plays a dominant role in

maintaining a functionally non-receptive uterine surface

with regard to blastocyst attachment.

It may inhibit implantation at the maternal cell

surface . (Journal.2.)

Mucin deficiency may enhance the chances of

lower quality embryos implanting successfully.

(Journal.2.)

Dating in the mid- to late-secretory phase relies on changes

in the endometrial stroma (3)

From Day 20, the stroma of the endometrium appears

oedematous. This is in response to prostaglandins (PG) E2

causing vascular permeability.

PGE2 stimulates capillary permeability either directly or

by means of increased histamine release which enhances

the appearance of oedema in the stroma. (Journal.2.)

Development of spiral arteries is a characteristic

feature of mid secretory phase(by days22-23of cycle)

and these vessels become increasingly coiled as their

length increases rapidly. (4)

During endometrial growth, spiral arteries lengthen at a

rate appreciably greater than the rate of increase in

endometrial tissue height or thickness. (3)

Specific angiogenic agents like members of VEGF family

produced from endometrial stroma cells and glandular

epithelium in response to estrogen and progesterone which

stimulates endothelial cell proliferation and increases

vascular permeability.(3 & 4)

Beginning 9 to 10 days after ovulation(Day 23-24),stromal

cells that surround the spiral arteries of the uterus enlarge

and develop eosinophilic cytoplasm, with a prominent Golgi

and endoplasmic reticulum. This process is referred to as

Pre decidualization(6)

Day 23 of the cycle the stroma contains well developed spiral arteries (arrows) surrounded by predecidual stromal Source: Fig6:Human Reproduction, Vol. 15, (Suppl. 1), pp. 119-128, 2000 (J.1)

Corkscrew appearance of glands with sacculated lumen: Source:Histology :A Text and Atlas :Michael H Ross : 6th ed/Figure23.16 B:Lippincott;Wolters kluwer

Laminin, fibronectin and type IV collagen surround

matrices of decidualizing cells. (6,4) .

Laminin, and fibronectin may provide a surface that

facilitates attachment of the embryo . (6)

Type IV collagen facilitates the attachment of

trophoblastic cells regulating the permeability and

nutrition of trophoblastic and endometrial cells. (journal-2)

The rounded decidual cells differentiate from spindle-

shaped fibroblast-like stromal cells under the influence

of progesterone.(6)

Peridecidual cells secrete numerous substances that

may be nutritious, metabolic or immunosuppressive like

insulin growth factor binding protein 1

(IGFBP1) ,prolactin and relaxin. (Journal.2.)

Multiple foci of these decidual cells spread throughout the

upper layer of the endometrium and form a dense layer

called zona compacta. This spreading is so extensive that

the glandular structures of the zona compacta become

inconspicuous. (6)

Stroma in the midzone of the functionalis shows little

predecidual changes,remains edematous hence appears

less opaque this is known as zona spongiosum here the

endometrial glands are more prominent (6)

The process of predecidualization is mediated by

Progesterone and its receptors .oestrogens do not

influence predecidualization . (Journal.2)

Evidence: ovariectomized women whose menstrual

cycle is induced by oestrogen and progesterone

administration present similar predecidualization

whether or not they receive oestrogens during the luteal

phase. (Journal.2.)

Figure showing Histologic organisation of secretory phase human endometrium .(Adapted from Reproductive Endocrinology Physiology ,PathoPhysiology and Clinical management; Yen,Jaffeand Barbieri ;4rth edition figure8-1)

Mitosis reappear in the stromal cells by day 26-27 as a

reflection of the decrease in progesterone activity which

marks a minor recrudescence of an estrogenic effect. (8)

In the Late premenstrual phase endometrium there is

infiltration of stroma by polymorphonuclear leukocytes . (3)

The endometrial stromal and epithelial cells produce

Interleukin-8(IL-8) ,a chemotactic /activating factor for

neutrophils which recruits neutrophils to the endometrium

just prior to the onset of menstruation (3)

Endometrium is capable of synthesizing Monocyte

chemotactic protein-1(MCP-1) chemoattractant for

monocytes. (3)

The rate of synthesis of IL-8 ,MCP-1 in the endometrial

stromal cells are modulated by Progesterone and

Transforming growth factor –β. (3)

In absence of fertilization, implantation and consequent lack of

sustaining quantities of HCG from the trophoblast , the otherwise

fixed lifespan of corpus luteum is completed ,and estrogen and

progesterone levels wane initiating the process of menstruation.(9)

Adapted from:-Di Fiore’s Atlas of Histology with Functional Correlations: Victor P. Eroschenko : Lippincott Williams & Wikkins : 12th Edition ;Chapter 21:Page 525-531

Table 2.3. Endometrial dating, secretory phase (SPL-1)

Interval phase: 14–15 d:No datable changes for 36–48

hours after ovulation

Early secretory phase: 16–20 d. Glandular changes predominate

16 d. Subnuclear vacuoles : Scattered small

irregular vacuoles can be caused by estrogen

alone.

17 d. Regular vacuolation—nuclei lined up with

subnuclear vacuoles

18 d. Vacuoles decreased in size . Early secretions in

lumen . Nucleus approaches base of cell.

19 d. Few vacuoles remain.Intraluminal secretion.

No pseudostratification, no mitoses

20 d. Peak of intraluminal secretions

Mid- to late secretory phase:21–27 d. Stromal changes predominate, variable secretory exhaustion

21 d. Marked stromal edema

22 d. Peak of stromal edema

23 d. Periarteriolar predecidual change . Spiral arteries

prominent

24 d. More prominent predecidual change .Stromal mitoses

recur.

25 d. Predecidual differentiation begins under surface

epithelium.Increased numbers of granular lymphocytes

26 d. Predecidua starts to become confluent.

27 d. Granular lymphocytes more numerous .Confluent sheets

of predecidua.Focal necrosis.

Why dating of endometrium? (SPL-1)

Routine histologic evaluation remains a cost-effective method of

determining the relative degree of endometrial development

through the menstrual cycle.

Dating of the endometrium yields information about :

(1) Histologic evidence of ovulation

(2) Dating the secretory phase gives a general assessment of

progesterone production by the corpus luteum and the ability of

the endometrium to respond to progesterone.

(3) presence or absence of endometrial abnormalities that may be

responsible for infertility.

Pitfalls of endometrium dating :(SPL-1)

1.Endometrium with surface epithelium is best for interpretation.

Absence of surface epithelium compromises the interpretation.

2. Tissue from the lower uterine segment or basalis is not

satisfactory for dating. Endometrium from these regions does not

respond fully to hormones.

3. Scattered subnuclear vacuoles in glands are not sufficient

evidence of ovulation. To be certain that ovulation has occurred,

more than 50% of the glands must show subnuclear vacuoles.

4. Focal cystic glands or nonreactive glands can occur in

normal endometrium and have no significance by themselves.

5. Compact predecidua with spindleshaped stromal cells may

not be appreciated as a true predecidual reaction. Directing

attention to stromal changes around spiral arteries assistsin

the identification of predecidua.

6.Lymphocytes and granular lymphocytes normally become

prominent in the stroma of the late secretory phase.These do

not represent inflammation.

7.The endometrium cannot be dated accurately when polyps,

inflammation, or other abnormalities are present.

8. several artifacts often complicate the histologic patterns. One

frequent artifact is tissue fragmentation caused by mechanical

disruption of the tissue. As a result, glands are detached from the

surrounding stroma, and fragmented glands become randomly

oriented.

As this traditional method of histological dating has low predictive

value further refinements have been employed in evaluation of

normal endometrium may evolve that have clinical utility.

Morphometric analysis has been attempted to increase the

accuracy of endometrial histologic dating.

Five morphometric measurements, including mitotic rate in

gland cells, amount of luminal secretion, volume fraction of

gland occupied by gland cell, amount of pseudostratification

of gland cells, and amount of predecidual reaction, added

precision to histologic dating

Immunohistochemical analysis for specific secretory products

of the endometrium.

ContinuATION OF The Endometrial Cycle

Synopsis : Proliferative changes occurs due to rise in level of ovarian

estrogens. Extends from 5th or 6th day to 14th day .

Secretory changes in the components are due to

combined effects of estrogen and progesterone liberated

from the corpus luteum after ovulation . Extends from 15th to

28th day of the cycle

Mitosis and growth is seen in Proliferative phase while

secretory changes and prepration of endometrium for

implantation is evident in secretory phase

WILLIAMS OBSTETRICS:21st edition ;4(69) :MC Grawhills publications

Endometrium growth regresses with the decline in

corpus luteum function compelling even greater coiling

of spiral arteries. (3)

Due to severe coiling of spiral arteries there is

increase in the resistance to the blood flow in these

vessels which is so marked that stasis develops

causing hypoxia of the endometrium (3&9)

Menstrual Phase

4 to 24 hours before bleeding into the endometrium there

is a period of intense vasoconstriction of spiral arteries at

the basal part. (3&4 )

Markee emphasized that intense vasoconstriction limits

the blood loss during menstruation. (3)

reduction in spiral artery blood flow and resultant stasis

before the time of vasoconstriction has been suggested as

primary cause of endometrial ischemia and tissue

degeneration. (3)

Endothelin -1 is a substance produced by endometrial

stromal cells in response to hypoxic endometrium which is a

potent vasoconstriction. (3)

Markee predicted that when an individual coiled artery

relaxed after a period of constriction , hemorrhage occurred

from that artery or its branches. (3)

This sequence of vasoconstriction ,relaxation and

hemorrhage appears to be well established but mechanisms

that actually brings about escape of blood from the vessels is

not certain. (3)

Menstruation is the result of enzymatic auto digestion and

ischemic necrosis. (Journal 2)

In the first part of the secretory phase, acid phosphatases

and lytic enzymes are confined to the lysosomes. Their release

is inhibited by Progesterone which stabilizes the lysosomal

membranes. (Journal 2 & 9)

with wanning of estrogen and progesterone levels, these

lysosomal membranes are disrupted and the enzymes are

released into the cytoplasm of epithelial , stromal and

endothelial cells (Journal 2 & 9)

These lytic enzymes digests their cellular elements,

leading to release of prostaglandins , extravasation of red

blood cells , tissue necrosis . (Journal2 & 9)

Invading leukocytes secrete enzymes that are members

of the matrix metalloproteinase (MMP) family which causes

the breakdown of the extracellular matrix and basal

membranes. As their action is localized, sloughing is

limited to the function of layer only. (Journal2 & 9& 3)

Haemostasis is the result of a balance between

coagulation and fibinolysis. (Journal 2)

Since progesterone maintains coagulation, any fall in

progesterone levels will give rise to fibrinolysis.(Journal 2) & 9)

Plasminogen activators are present in the menstrual

endometrium and originate from the endometrial vascular

endothelium. They convert plasminogen into plasmin

which in turn prevents the menstrual blood from clotting.

(Journal 2 )

Fibrinolysis increases progessively, leading to

menstrual bleeding. (Journal 2 & 9)

Vasomotor phenomena bring about vasoconstriction

and vascular relaxation. They are regulated by

Prostaglandin F2α which increase during the secretory

phase and reach peak levels during the menstrual

phase. (Journal 2 & 9)

PGF cause vasoconstriction of the basal arteries

which gives rise to circulatory arrest in the spiral arteries

and contraction of the myometrium at the boundary

between endometrium and myometrium. (Journal 2 & 9)

These tissue and vascular phenomena together lead

to tissue desquamation or sloughing and the shedding of

menstrual blood. (Journal 2 & 9)

Blaustein's Pathology of the Female Genital Tract :Ancel Blaustein, Robert J. Kurman

Fig.2(E) The white arrow indicating the hysteroscopic appearance of an unshed endometrium adjacent to a zone of shed surface epithelium, demonstrating the piecemeal nature of endometrial shedding (black arrows indicate multiple fine tubes) on Day 1 of menstruation. Adapted from: Garry et al : Human Reproduction, Vol.1, No.1 pp. 1–9, 2009

Adapted from:-Di Fiore’s Atlas of Histology with Functional Correlations: Victor P. Eroschenko : Lippincott Williams & Wikkins : 12th Edition ;Chapter 21:Page 525-531

Source :BEST & TAYLOR’S :The Physiological Basis of Medical Practice, 13th Edn/968;Lippincott williams and wilkins.

Regenerative Phase

Starts at the moment of menstruation and constitutes

the first few days of the proliferative phase. (Journal 2)

Tissue necrosis provokes a marked inflammatory

response with the production of pro-inflammatory,

chemotactic and anti-inflammatory cytokines. (Journal 2)

Regeneration also depends on macrophages and

polymorphonucleacytes which help to clean up the necrotic

area. (Journal 2)

The stromal cells of the basal component of the

mucosa proliferate to replace the shed endometrium and

subsequently are active in re-establishing the

endometrium. (Journal 2)

This synthesis is associated with a regeneration of the

epithelium with gland proliferation, starting from the

basal component and in the surface epithelium

around the tubes and isthmus. (Journal 2 & 10)

This post-menstrual regenerated epithelium binds to the

fibroblasts of the underlying stroma, with the stromal cells

forming clumps onto which the surface epithelium can

migrate . (Journal 2 & 10)

Epithelial growth may in fact be stimulated by the

underlying fibroblast. (Journal 2 & 10)

Tenascin, a fibronectin-inhibitor synthesized by the

fibroblasts of the endometrial stroma, may also play a role

in facilitating epithelial migration (Journal 2 & 10)

(C) Some of the tubes are the residual stumps of endometrial glands. Here, a high-power SEM image of an isolated endometrial glandular stump in material left after Day 1 menstrual shedding. The black arrows indicate characteristic ciliated cells interspersed between the epithelial cells.

(F) A high-power SEM image of a Day 1 surface appearance of the basalis surfaceshowing a fibrinous matrix in which numerous red blood cells are adherent. Macrophages are also seen (black arrows).ADAPTED from: Garry et al : Human Reproduction, Vol.1, No.1 pp. 1–9, 2009

(C) (F)

A remarkable feature seen in human females and

rhesus monkey endometrium in absence of pregnancy is

its regeneration on a cyclic basis attributed to a unique

stem cell population and its progenitor cells confined to

the basalis layer and belived to initiate and sustain the

process of regeneration(11)

.

.

The role of oestradiol in endometrial regeneration is only

evident after the necrotic area has been cleaned up.

(Journal2 )

Oestradiol serum titres are very low during the

menstrual phase and regeneration seems due initially just

to a repair mechanism. (Journal2 )

Evidence: Endometrial regeneration in ovariectomized

rabbits is identical to that of rabbits with normal ovaries

(Journal2 & 10)

Post menstrual endometrial remodelling is induced by

prostaglandins released following tissue breakdown and

there is no role of hormones with serum estrogen and

ER ,PR receptors as low as premenstrual values. ( 11)

Day 7-12 serum estrogens and ER ,PR concentrations

increase resulting in marked increase in DNA synthesis

and mitotic activity in endometrial cells (10)

VEGF (Vascular endothelial growth factor) produced

by endometrium in response to estrogen and hypoxia

plays an important role in angiogenesis and endometrial

repair. (2)

By the 5th day of cycle surface epithelium cuboidal in

nature is derived from gland lumina and stromal cells.

and revascularization of the endometrium is in progress. (1)

Two anatomical parameters have been sugested for the

evaluation of the endometrium by ultrasound (SPL-2) :

Endometrial Pattern

Endometrial Thickness

Proliferative endometrium. Three lines representing the basal endometrium (red arrows) and central endometrial cavity echo (yellow arrow)

2 hypoechoic areas between the 3 sharp lines. The central echo denotes the cavity and the line of contact between the two endometrial surfaces.

SOURCE :2ND USG Plate : Narendra Malhotra et al :Rational Use of TVS/Color and 3D in Evaluating Subfertile Women : Donald School Journal of Ultrasound in Obstetrics and Gynecology, July-September 2011;5(3):273-287.

Proliferative Endometrium

2ND

USG Plate

1st USG Plate

3rd USG Plate

Secretory endometrium demarcated between the two arrows. The basal endometrium is no longer seen.

Only the echogenic endometrium is measured (line). The surrounding sonolucent halo (arrows) should not be included in the measurement.

SOURCE: 3rd ,4rth ,5th USG Plates :Ultrasound Educational Press : Peter W. Callen, M.D. Professor of Radiology, Obstetrics, Gynecology and Reproductive Science University of California Medical Center, San Francisco, California .

Secretory Endometrium

4rth USG Plate

5th USG Plate

Endometrial Thickness and Menstrual Cycle USG FINDINGS

ADAPTED FROM : Narendra Malhotra et al :Rational Use of TVS/Color and 3D in Evaluating Subfertile Women : Donald School Journal of Ultrasound in Obstetrics and Gynecology, July-September 2011;5(3):273-287.

What’s the need to study endometrial pattern and endometrial thickness ?

There is various conflicting controversial observations

and no generalized consensus that the measurement of

endometrial thickness have predictive value for pregnancy

or not . (SPL-2)

However it has been suggested that the main advantage

of measuring endometrial thickness lies in its high

negative predictive values in cases where there is

minimal endometrial thickness. (SPL-2)

It has been observed that thin endometrium (<7mm) and thick

endometrium (>14mm) are frequently associated with

miscarriages. (SPL-2)

Sundstrom described a successful pregnancy in a patient

who’s endometrium measured only 4mm. (SPL-2)

Also poor endometrial pattern does not exclude pregnancy. (SPL-2)

Thus, endometrial pattern can serve as prognostic value in

both fresh IVF and frozen embryo transfer cycles as endometrial

pattern is not influenced by type of ovarian stimulation. (SPL-2)

REPRODUCTIVE ASSISTED FERTILIZATION

Assisted reproductive technologies  is used for treatment

of infertility.

First successful birth of a "test tube baby", Louise brown

occurred in 1978 born at Oldham General Hospital by planned

Caesarean section delivered by John Webster.

Robert G Edwards the Physiologist who developed the

treatment was awarded the Nobel Prize in Physiology or Medicine

in 2010

Adrina Iliescu held the record of oldest women to give birth

using IVF and donated egg when she gave birth in 2004 at age of

66 .

Subhash Mukhopadhayay ;a Reproductive

Physiologist who created world’s 2nd and India’s 1st child using

in vitro fertilization Durga(Kanupriya Agarwal ) born 67 days

after 1st IVF baby in UK.

Thanks to TC Anand kumar to bring out this

information to the world .

Assisted Reproductive Technology (ART) includes (12)

In vitro fertilization-embryo transfer (IVF-ET)

gamete intrafallopian transfer (GIFT)

zygote intrafallopian transfer (ZIFT)

frozen embryo transfer (FET)

Approximately 99 percent of ART cycles performed are IVF-ET

The basic steps in an IVF treatment cycle are (12)

Ovarian stimulation Egg retrieval Fertilization Embryo culture, Embryo transfer.

Ovarian Stimulation (12)

During ovarian stimulation or ovulation induction, medications or

“fertility drugs,” are used to stimulate multiple eggs to grow in the

ovaries rather than the single egg that normally develops each

month .

Multiple eggs are stimulated because some eggs will not

fertilize or develop normally after fertilization.

TABLE Medications for Ovarian Stimulation• Human menopausal gonadotropin (hMG) • Follicle stimulating hormone (FSH) • Luteinizing hormone (LH) (used in conjunction with FSH)• Human chorionic gonadotropin (hCG) • Clomiphene citrateMedications to Prevent Premature Ovulation• Gonadatropin releasing hormone (GnRH) agonists• GnRH antagonists

Clomiphene citrate are administered orally while the other medications listed are given by injection.

SOURCE: AMERICAN SOCIETY FOR REPRODUCTIVE MEDICINE 2011 : ASSISTED REPRODUCTIVE TECHNOLOGIES :A Guide for Patients

The ovaries are evaluated during treatment with

vaginal ultrasound examinations to monitor the

development of ovarian follicles . (12)

Blood samples are drawn to measure the response to

ovarian stimulation medications. (12)

Normally, estrogen levels increase as the follicles

develop, and progesterone levels are low until after

ovulation (12)

Ovarian follicles, stimulated by ovulation medications, visible on ultrasound. The dark, circular areas are the follicles.

When the follicles are ready, hCG are given. (12)

HCG replaces the woman’s natural LH surge and causes the

final stage of egg maturation so the eggs are capable of being

fertilized. (12)

The eggs are retrieved before ovulation occurs, usually 34 to 36

hours after the hCG injection is given. (12)

Egg Retrieval

Egg retrieval is usually accomplished TVS aspiration

performed in the OPD. (12)

Laparoscopy may be used to retrieve the eggs using a

small telescope placed in the umbilicus. (12)

Fertilization and Embryo Culture

After the eggs are retrieved, they are examined in the

laboratory for maturity and quality. Mature eggs are placed

in an IVF culture medium and transferred to an incubator to

await fertilization by the sperm. (12)

Sperm may be obtained from the testicle, epididymis, or vas

deferens or by manstrubation . (12)

Fertilization may be accomplished by insemination, where motile

sperm are placed together with the oocytes and incubated

overnight or by intracytoplasmic sperm injection (ICSI), where a

single sperm is directly injected into each mature egg (12)

Mature unfertilized egg Intracytoplasmic sperm injection (ICSI),

A fertilized egg has divided once and is now a two-cell embryo.

A fertilized egg has divided once and is now a eight -cell embryo.

Blastocyst

By the third day, a normally developing embryo will

contain approximately 6 to 10 cells. (12)

By the fifth day, a fluid cavity forms in the embryo, and

the placenta and fetal tissues begin to separate. An

embryo at this stage is called a blastocyst. (12)

Embryos may be transferred to the uterus at any time

between one and six days after the egg retrieval. (12)

If successful development continues in the uterus, the

embryo hatches from the surrounding zona pellucida

and implants into the lining of the uterus approximately 6

to 10 days after the egg retrieval. (12)

Assisted hatching (AH) is a micromanipulation

procedure in which a hole is made in the zona pellucida

just prior to embryo transfer to facilitate hatching of the

embryo. (12)

Preimplantation genetic diagnosis (PGD) is performed at

some centers to screen for inherited diseases (12)

Embryos that do not have the gene associated with the

disease are selected for transfer to the uterus. (12)

Embryo Transfer

The next step in the IVF process is the embryo transfer. (12)

No anesthesia is necessary, although some women may

wish to have a mild sedative. (12)

One or more embryos suspended in a drop of

culture medium are drawn into a transfer catheter, a

long, thin sterile tube with a syringe on one end. (12)

The physician gently guides the tip of the transfer

catheter through the cervix and places

the fluid containing the embryos into the uterine cavity (12)

The physician identifies the cervix using a vaginal

speculum . (12)

EMBRYO TRANSFER THROUGH CERVIX

CLINICAL IMPORTANCE OF ENDOMETRIUM :

The implantation potential of good quality embryos remains low

during IVF/ET treatment, despite advances in ovarian stimulation

regimens, the method of assisted fertilization and improved culture

conditions.

Successful implantation depends on a close dialog between the

blastocyst and the receptive endometrium.

Ultrasound parameters including endometrial thickness, endometrial

pattern, endometrial volume, Doppler study of uterine arteries and

endometrial blood flow have been used to assess endometrial

receptivity during IVF treatment.(SOURCE:Predictive value of endometrial thickness, pattern and sub-endometrial blood flows on the day of hCG by 2D doppler in  in-

vitro fertilization cycles: A prospective clinical study from a tertiary care unit Neeta Singh , et al J Hum Reprod Sci. 2011 Jan-Apr; 4(1):

29–33.)

SOURCE: Endometrial receptivity and scoring of predictions for implantation and new markers : Narendra Malhotra et al :Dorland school journal of ultrasound in obstetrics and gynaecology:october-december:2010 4(4);439-446

Perfect score of 20 has been associated with conception 100 % of the time with this system but in limited experience.

Attempt to Increase endometrial

thickness………………

Endometrial growth is thought to depend on uterine artery

flow and endometrial development has great importance on

in-vitro fertilization.

Nitric oxide relaxes vascular smooth muscles through c-

GMP mediated pathway and NO Synthase isoforms have

been identified in the uterus.

Phosphodiesterase hydrolyse c-AMP and c-GMP.

Sildenafil citrate (viagra) a type 5 specific

Phosphodiesterase inhibiting aguments the vasodilatory

effects NO by preventing degradation of c GMP.

Pulsatile index was decreased in 7 days after

sildenafil indicating increased blood flow.

Combination therapy of Sildenafil and oestradiol

valerate improved blood flow and endometrial thickness in

all patients as per below mentioned journal next slide .

NTG have been successfully used in improving

uterine blood flow and endometrial lining but with

increased side effects like hypotension and headaches.

Study on effects of sildenafil (vaginal suppositories) in

increasing uterine bleeding and endometrial thickness is

in preliminary phase and needs more intensive studies.

Source:Vaginal sildenafil(viagra):A preliminary report of a novel method to improve uterine artery blood flow and endometrial development in patients undergoing IVF :Geoffrey Sher et al :Human Reproduction :vol 15 no.4 pp.806-809 ,2000

NEWER Modality to increase endometrial thicknessG-CSF has also been employed in the proliferation of the

thin endometrium which fails to respond with standard therapy.

Evidence:1. Gleicher  et al., perfused G-CSF transvaginally into

the uterus of four patients undergoing IVF with thin endometrium

after standard endometrial preparation.

This resulted in 7 mm of additional endometrial proliferation which

was previously resistant to estrogen and vasodilators therapy and

all the patients successfully underwent Endometrial Thickness and

conceived.

This demonstrates G-CSF as an innovative

remedy in patients with unresponsive,

inadequate, and thin endometrium.(Journal.8)

Evidence:2: Gleicher  et al :described in 21 consecutive infertile

women with endometria <7 mm on the day of hCG administration

in their first IVF cycles. All previous cycles using traditional

treatments with estradiol, sildenafil citrate (Viagra™) and/or beta-

blockers had been unsuccessful.

G-CSF (Nupogen™) was administered per intrauterine catheter

by slow infusion before noon on the day of hCG administration.

Result: This cohort study is supportive of the effectiveness of G-

CSF in expanding chronically unresponsive endometria.

(Journal.9.)

Evidence:3: Pratap Kumar et al Evaluated the success rate of

IVF with supplementation of G-CSF in embryo culture media and

also the success of women undergoing IVF with thin endometrium

which fails to proliferate with standard treatment, but respond with

transvaginal instillation of G-CSF into the endometrium.

Results were remarkable as the survival rate of the embryo

cultured with G-CSF till 12th week and birth were significantly

higher which proves the efficacy of the G-CSF in assisted

reproduction technology.(Journal .10.)

Dysfunctional uterine bleeding (DUB)

defined as abnormal, irregular bleeding (excessively heavy,

prolonged, or frequent intervals of bleeding) in the absence

of demonstrable pelvic disease, complications of pregnancy

or systemic disease.

The exact mechanism is uncertain but is thought to be

caused by dysfunction of hypothalamic-pituitary-ovarian axis.

SOURCE: Management of dysfunctional uterine bleeding based on endometrial thickness: Ozgul Muneyyirci-Delale et al :International Journal of Women’s Health 2010:2 297–30

TVS is an excellent non-invasive tool to diagnose anatomic causes of dysfunctional uterine bleeding.

Getpook et al showed that ET of 8 mm or less is unlikely to be associated with malignant pathology.

Management of DUB based on endometrial thickness is an effective approach to control acute uterine bleeding.

SOURCE: Management of dysfunctional uterine bleeding based on endometrial

thickness: Ozgul Muneyyirci-Delale et al :International Journal of Women’s

Health 2010:2 297–30

ADAPTED FROM: Management of dysfunctional uterine bleeding based on endometrialthickness: Ozgul Muneyyirci-Delale et al :International Journal of Women’s Health 2010:2 297–302

THE ENDOMETRIAL CYCLE

A CASE REPORT BY: Nagori.et al Done at: Dr.Nagori Institute

for Infertility Published at: J Hum Reprod Sci. 2011 Jan-Apr; 4(1): 43–48 (Journal.11.)

In a woman with severe Asherman's syndrome, curettage

followed by placement of intrauterine contraceptive device

(IUCD) (IUCD with cyclical hormonal therapy) was tried for 6

months, for development of the endometrium.

When this failed, autologous stem cells were tried as an

alternative therapy.

From adult autologous stem cells isolated from patient's

own bone marrow, endometrial angiogenic stem cells were

separated using immunomagnetic isolation.

These cells were placed in the endometrial cavity

under ultrasound guidance after curettage.

Patient was then given cyclical hormonal therapy.

Endometrium was assessed intermittently on

ultrasound. On development of endometrium with a

thickness of 8 mm and good vascularity, in

vitro fertilization and embryo transfer was done.

This resulted in positive biochemical pregnancy

followed by confirmation of gestational sac, yolk sac,

and embryonic pole with cardiac activity on ultrasound.

Endometrial angiogenic stem cells isolated from

autologous adult stem cells could regenerate injured

endometrium not responding to conventional treatment

for Asherman's syndrome.

1.Dutta’s textbook of gynaecology;chap7/83

2.Jeffcoate’s Gynecology 7th ed/79

3.Williams Obstetrics21st ed/66-76 ; 23rded/chap 3

4.Best & Taylor Physiology 13th ed/968-69

5.Physiology of reproduction ,Odell& moyer /35-36

6.Boron Physiology 2006/1161-62

7.Medical Physiology:Rodeney A

Rhodes :4thed:Lippincott:chap37/703

8.Hanes&Taylor:Obsterics & Gynaecology Pathology :Churchill

Livingstone :Vol.1/5th ed/391-409

9.Clinical Gynecology ,Endocrinology and Infertility:Leon

Speroff :Wolters

& Lippincott :7th ed / chap 4/115-131

10.Blaustein's Pathology of the Female Genital Tract,; A.Blaustein,

R.Kurman:Springer:5th ed/421-461

11.Knobil and Neill's Physiology of Reproduction:J.Neill :

Elsevier academic Press  , 3rd Edition :vol.1/chap.9/368

Textbook Reference

12.Handbook on ASSISTED REPRODUCTIVE TECHNOLOGIES A guide for

patients :revised 2011:Pg:1-27:AMERICAN SOCIETY FOR REPRODUCTIVE

MEDICINE.

SPL-1: Mazur MT, Kurman RJ. Normal endometrium and infertility evaluation.

In: Mazur MT, Kurman RJ, editors. Diagnosis of endometrial biopsies and

curettings: A practical approach. 2 nd ed. New York: Springer Verlag; 2005. p.

7-33.

SPL-2: David K Gardner : Textbook of Assisted Reproductive Technologies .3rd

edn/648-652

Atlas: Di Fiore’s Atlas of Histology with Functional Correlations:

Victor P. Eroschenko : Lippincott Williams & Wikkins : 12th

Edition ;Chapter 21:Page 525-531

Atlas: Histology :A Text and Atlas :Michael H Ross : 6th ed/Figure23.16

B:Lippincott;Wolters kluwer

Journal.2. : Christine Bergeron: Morphological changes and protein

secretion induced by progesterone in the endometrium during the luteal

phase in preparation for nidation : Human Reproduction, Vol. 15, (Suppl.

1), pp. 119-128, 2000 .

Journal.3. : Garry et al : A re-appraisal of the morphological changes

within the endometrium during menstruation: a hysteroscopic,

histological and scanning electron microscopic study : Human

Reproduction, Vol.1, No.1 pp. 1–9, 2009

Journal References

Journal.1:I.Mylonas et al :Steroid receptors ERα, ERß, PR-A and PR-B are

differentially expressed in normal and atrophic human

endometrium :Histol Histopathol (2007) 22: 169-176

Journal.5. :Neeta Singh et al :Predictive value of endometrial thickness,

pattern and sub-endometrial blood flows on the day of hCG by 2D doppler

in in-vitro fertilization cycles: A prospective clinical study from a tertiary

care unit : J Hum Reprod Sci. 2011 Jan-Apr; 4(1): 29–33.

Journal.6. :Narendra Malhotra et al :Endometrial receptivity and scoring

of predictions for implantation and new markers : Dorland school journal

of ultrasound in obstetrics and gynaecology:october-december:2010

4(4);439-446

Journal.4. :Narendra Malhotra et al :Rational Use of TVS/Color and 3D in

Evaluating Subfertile Women : Donald School Journal of Ultrasound in

Obstetrics and Gynecology, July-September 2011;5(3):273-28

Journal.9. Gleicher N,Vidali A ,Barad DH: Successful treatment of

unresponsive thin endometrium: Fertil Steril. 2011 May;95(6):2123.e13-7.

Journal.8. Ozgul Muneyyirci-Delale et al :Management of dysfunctional

uterine bleeding based on endometrial thickness: :International Journal

of Women’s Health 2010:2 297–30

Journal.7.: Geoffrey Sher et al :Vaginal sildenafil(viagra):A preliminary

report of a novel method to improve uterine artery blood flow and

endometrial development in patients undergoing IVF:Human

Reproduction :2000 :vol 15 no.4 pp.806-809

 

Journal.10. Gleicher N et al :A pilot cohort study of granulocyte colony-

stimulating factor in the treatment of unresponsive thin endometrium

resistant to standard therapies. Hum Reprod 2013 Jan;28(1):172-7

Journal.11. Pratap Kumar and Siddharth Mahajan : Preimplantation and

postimplantation therapy for the treatment of reproductive failure:J Hum

Reprod Sci. 2013 Apr-Jun; 6(2): 88–92

Journal.12. Nagori CB et al Endometrial regeneration using autologous adult

stem cells followed by conception by in vitro fertilization in a patient of severe

Asherman's syndrome:J Hum Reprod Sci 2011 Jan;4(1):43-8

THANKS FOR PATIENT LISTENING