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WELCOME

INTRODUCTIONThe epithelia of the female genital tract are sensitive to estrogen , progesterone , androgens & various additional steroid hormone derivatives & metabolites.In general estrogen promotes & progesterone inhibits squamous cell maturation.Hormones may influence the morphology staining characteristics of the cervical , endometrial , vaginal , urethral & bladder mucosa cell. Hormonal evaluation is based on the degree of maturation & glycogen storage of the exfoliated squamous cells. For useful cytologic evaluation , some information about the age , menstrual history & previous hormonal , surgical or treatment of the patient should accompany the smear.

INDICATIONS FOR CYTOLOGIC HORMONAL EVALUATION1)Assessment of ovarian function.

During puberty & reproductive , menopausal & post menopausal years after hysterectomy.During menstrual disorders.In premature menses (childhood).To determine ovulation time.

2)Assessment of abnormal hormonal production (placenta, ovaries & other endocrine organs).

Before , during & after a pregnancy (fertility study , threatened abortion , retained placenta ).

Existence of functioning (hormone producing) tumors.

Various endocrine disorders.

Assessment & guidance for hormonal therapy.

NORMAL HORMONAL PATTERNSESTROGEN

It increases cellular maturation & proliferation of all layers of squamous epithelium mostly Superficial cells and Intermediate cells.

DECREASING LEVEL

Deficiency of estrogen increases parabasal cells.

The mucosa becomes thin & superficial layer is absent in deficient estrogen stimulation.

INCREASING LEVEL

Increase in estrogen level enlarges the parabasal cells to intermediate .this continue to enlarge & mature into keratinized superficial cells.

Increases the amount of intra cellular glycogen ( by protein & nucleic acid synthesis )

High estrogenic effect in a vaginal smear is recognized by a clean background & increased eosinophilic & karyopyknotic indices. PROGESTERONEIt produce proliferation of intermediate squamous epithelium at the time of exfoliation. INCREASING LEVELClusters of intermediate cells dominate the smear.The amount of intracellular glycogen-increasesNo. of leukocytes decreases

ANDROGEN

Male sex hormone.

Small amount is present in female.

INCREASING LEVEL It increases the no. of exfoliated large parabasal cells with central , almost pyknotic nuclei & dense , glycogen containing cytoplasm.GUIDELINES FOR SPECIMEN COLLECTION & SUITABLE FOR HORMONAL ASSESSMENT

1)SPECIMEN COLLECTION GUIDELINES:Smear taken from the lateral vaginal wall Smear taken from the junction of the middle & upper 1/3rd of the vagina.2)FACTORS THAT MAKE A SMEAR UNSUITABLE FOR HORMONAL ASSESSMENT:Material not collected properly.Adequate clinical information not provided (age , menstrual status, treatment etc)Evidence of an infection.Inflammation. Air-drying artifact.

HORMONAL PATTERN AT DIFFERENT STAGES OF LIFELIFE STAGEHORMONAL PATTERNNewbornSterileNo inflammationIntermediate cells superficial cells1st - 2 week of lifeAcquires bacteriaNeutrophils appearAtrophic cellsChildhoodMaturation & cyclic alternations occur months (or)years before menarche, due to cycles that are initially anovulatoryCyclic women , proliferative phaseIncreasing squamous cell maturationCyclic women , post ovulatory phaseDecreasing squamous cell maturation PregnancyNavicular cells may be numerousLactationAtrophy persists but some maturation may appear slowly .Post partumAtrophyMenopauseAtrophy (or) intermediate cell predominate12CELLS1) SUPERFICIAL CELLSCell flat , delicate & transparent.

Size 35-45 micro meter in diameter.

Nuclei small , dark & pyknotic (nuclear material become condensed & shrunken.

Cytoplasm delicate pink (eosinophilic / acidophilic)2) INTERMEDIATE SQAMOUS CELLSCell flat Size 35-45 micro meterNucleus spherical / oval -vesicularCytoplasm basophilicA variant of intermediate cells boat shaped navicular cells.

Navicular cells common in early menopause & pregnancy.Oval shaped cells store glycogen in the form of cytoplasmic deposits stains yellow in PAP stain PARABASAL CELLS

Size 12 -30 micro meter.

Nuclei vesicular.

Shape round / oval.

Cytoplasm basophilic.4) BASAL CELLSShape round / oval.

Cytoplasm scanty , basophilic.

Nuclei vesicular -fine chromatic granules -occasionally tiny round nucleoliCYTOLOGICAL INDICESThe degree of proliferation , maturation & desquamation of vaginal cells is influenced by various hormones.

The cytological assessment of these hormones is given by different types of indices . All are based on recognition & exact typing of the epithelial cells exfoliated from the surface of the stratified squamous vaginal mucosa .If the smear is to inflammatory & a repeat specimen is not possible after the proper treatment , the exfoliated urethral cells in the first portion of a voided urine specimen can be examined for this evaluation.

The terminal part of the urethral mucosa is almost as sensitive to hormonal changes as in the vaginal mucosa but more difficult to evaluate.TYPES OF INDICESMaturation index (MI)It is determined by the percentage of the basal , intermediate & superficial cells as presented as 3 part ratio with the basal cells started 1st , intermediate cells 2nd & superficial cells 3rd. Example : MI = 80 /20 / 0 Indicates : 80 % - Parabasal cells 20 % - Intermediate cells 0 % - Superficial cells

RESULTS READ AS:Basal Intermediate superficial 0 0 100 Shift - left 0 100 0 Shift -middle 100 0 0 Shift right.RESULT READ AS

Shift to left atrophic effect.

Shift to right increased estrogen like effect.

Shift to midzone progesterone like effect

2)KARYOPYKNOTIC (KPI) / CORNIFICATION INDEX

The percentage of squamous cell with sharp squared cytoplasmic edges with pyknotic nuclei compared with other mature squamous cells with vesicular nucleus.

Parabasal cells are not counted.3)MATURATION VALUE (MV) This method is useful for providing hormonal evaluation data to a computer.

Each parabasal cell is counted as 0 Intermediate cell as 0.5 superficial cell as - 1.0 The addition of all the values given to the first 100 epithelial cells is recorded. Example : MI =0 / 35 / 65 0 x 0 =0 35x 0.5 = 17.5 65x1.0 = 65.0 MV = 82.5 MV of 100 indicates pure population of superficial squamous cells MV of 0 indicates pure population of parabasal cells. STAGES MI MV VARIATIONNewborn 0 / 90 / 10 55 +/ - 10Infancy 80 / 20 / 0 10 + / - 20Pre ovulatory 0 / 40 / 60 80 + / - 10Post ovulatory 0 / 70 / 30 65 + / - 15Menopause 0 / 80 / 20 60 + / - 20Post menopause 50 / 50 / 0 25 + / - 40Estrogen therapy 0 / 10 / 90 95 + / - 10Progesterone therapy 0 / 90 / 10 55 + / - 10Androgen therapy 20 / 80 / 0 40 + / - 104)EOSINOPHILIC INDEX (EI) The no. of mature squamous cells with eosinophilic (pink) cytoplasm , nuclear appearance is compared with the no. of other mature squamous cells with basophilic (blue) cytoplasm. The result is given in ratio. Parabasal cells not counted.

Example : 2:14 2 eosinophilic sq. cell 14 basophilic sq. cells5)FOLDED CELL INDEX (FCI) No . of mature sq. cells with folded cytoplasmic rims are compared with no. of mature cells with flat non folded cytoplasm.

The folded cells with cytoplasmic glycogen are considered as less mature than flat cells.6)SUPERFICIAL CELLS INDEX (SCI)

Superficial sq. cells with any type of nucleus is compared with any other sq. cell present in the smear.

7)CROWED CELL INDEX (CCI)

The no. of mature sq. cells that are clustered together in groups of 5 or 6 cells is compared to the no. of similar cells found singly / less than 3 or 4. INDICES USED FOR HORMONAL ASSESSMENTTYPE OF INDEXCELL TYPE RATIO COMPAREDMaturation indexParabasal : intermediate : superficialKaryopyknotic indexSuperficial : intermediateEosinophilic indexEosinophilic : basophilicFolded cell indexFolded cytoplasm : non folded cytoplasmCrowed cell indexClusters of more than 4 cells : single cellNORMAL CYTOHORMONAL AVERAGES1)NEW BORN (UPTO 8 WEEKS) MI =0 / 90 / 10 Intermediate cell increase with glycogen in their cytoplasm. - this is due to maternal hormones in infants blood. 1st 4 8 days - Very little cellular debris , bacteria , leucocytes , lymphocytes (or)mucous can be found. 2 3 weeks increased no. of leucocytes & micro organism occurs. 3 4 weeks intermediate cell decreases. 2) INFANCY & NEWBORN (3 WEEKS TO PUBERTY)

MI = 80 / 20/ /0

Vaginal pool smears are scanty in cells & contain mainly parabasal cells.

Leucocytes few.

These smears are very similar to the cells found in the late postmenopausal period except for their smaller size & the absence of inflammatory background.3)MENSTRUAL AGE (REPRODUCTIVE PERIOD)

MI = 0 / 70 / 30

The vaginal cellular changes start to appear several months before the beginning of menstruation. A nucleated eosinophilic sq. cells are present.

Basal cells & polymorphonuclear cells are the main cells found during the reminder of the cycle.

Superficial & intermediate cells always present. Parabasal cells-few. 4)DURING PRE OVULATORY TIME.(Proliferative phase, 3-14 day of the cycle)

MI=0/40/60

8th day of cycle -the cyanophilic intermediate cells gradually increases in size with progressive cytoplasmic eosinophilia & nuclear pyknosis. - no. of leukocytes decreases. mucous decreases. 5) DURING OVULATION & POST OVULATORY TIME (Secretory phase 15 19th days)

MI = 0 / 70 / 30

Super ficial & eosinophilic cells decreases

Intermediate cells increases & the cells show a tendency to cluster together.

6) DURING MENSTRUAL FLOW.

MI = 0 / 60 / 40 Increase of fresh & old red blood cells , cyanophilic intermediate cells, histiocytes, mucous & leukocytes.

Grandular & stromal endometrial cells are seen singly in clusters /in acinic formation.

The evaluation of smears taken at 3 day intervals will indicate if ovulation has occurred & the level of estrogenic effect. 7) MENOPAUSE

MI = 0 / 80 / 20

Exfoliated superficial & intermediate sq. cells become smaller & show some decrease in their staining capacity. 8) EARLY POST MENOPAUSE

2-6 years cessation of menstruation a decrease in estrogenic activity occurs.

Parabasal & intermediate cells increases

Glycogen is found in some of the cells.

9) LATE POST MENOPAUSE

MI = 100 / 0 / 0

Some smear may become completely atrophic.

Atrophic parabasal cells: size varies shape oval / round with mild irregularities cytoplasm cyanophilic -show degenerative changes in the form of vacuolization. No glycogen is present.

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