INTRODUCTION The 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 EVALUATION 1)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 PATTERNS  ESTROGEN 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.

 PROGESTERONE It produce proliferation of intermediate squamous epithelium at the time of exfoliation. INCREASING LEVEL Clusters of intermediate cells dominate the smear. The amount of intracellular glycogen-increases No. 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 1/3 rd 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 LIFE LIFE STAGEHORMONAL PATTERN Newborn Sterile No inflammation Intermediate cells superficial cells 1 st - 2 week of life Acquires bacteria Neutrophils appear Atrophic cells Childhood Maturation & cyclic alternations occur months (or)years before menarche, due to cycles that are initially anovulatory Cyclic women, proliferative phase Increasing squamous cell maturation Cyclic women, post ovulatory phase Decreasing squamous cell maturation

PregnancyNavicular cells may be numerous LactationAtrophy persists but some maturation may appear slowly. Post partumAtrophy MenopauseAtrophy (or) intermediate cell predominate

CELLS 1) SUPERFICIAL CELLS Cell – flat, delicate & transparent. Size – micro meter in diameter. Nuclei – small, dark & pyknotic (nuclear material become condensed & shrunken. Cytoplasm – delicate pink (eosinophilic / acidophilic)

2) INTERMEDIATE SQAMOUS CELLS Cell – flat Size – micro meter Nucleus – spherical / oval -vesicular Cytoplasm – basophilic A 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

3) PARABASAL CELLS Size – micro meter. Nuclei – vesicular. Shape – round / oval. Cytoplasm – basophilic.

4) BASAL CELLS Shape – round / oval. Cytoplasm – scanty, basophilic. Nuclei – vesicular -fine chromatic granules -occasionally tiny round nucleoli

CYTOLOGICAL INDICES The 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 INDICES Maturation 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 1 st, intermediate cells 2 nd & superficial cells 3 rd. Example : MI = 80 /20 / 0 Indicates : 80 % - Parabasal cells 20 % - Intermediate cells 0 % - Superficial cells

RESULTS READ AS: Basal Intermediate superficial Shift - left Shift -middle 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 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 = x1.0 = 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 VARIATION Newborn 0 / 90 / / - 10 Infancy 80 / 20 / / - 20 Pre ovulatory 0 / 40 / / - 10 Post ovulatory 0 / 70 / / - 15 Menopause 0 / 80 / / - 20 Post menopause 50 / 50 / / - 40 Estrogen therapy 0 / 10 / / - 10 Progesterone therapy 0 / 90 / / - 10 Androgen therapy 20 / 80 / / - 10

4)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. cells

5)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 ASSESSMENT TYPE OF INDEXCELL TYPE RATIO COMPARED Maturation indexParabasal : intermediate : superficial Karyopyknotic index Superficial : intermediate Eosinophilic indexEosinophilic : basophilic Folded cell indexFolded cytoplasm : non folded cytoplasm Crowed cell indexClusters of more than 4 cells : single cell

NORMAL CYTOHORMONAL AVERAGES 1)NEW BORN (UPTO 8 WEEKS) MI =0 / 90 / 10 Intermediate cell – increase with glycogen in their cytoplasm. - this is due to maternal hormones in infant’s blood. 1 st 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 8 th 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.