Microscopic study on the total number of follicles of ovary in Bangladeshi female cadavers

Context: The ovaries are paired female reproductive and endocrine glands. It is an unique organ in the female body. It undergoes various changes under the influence of hormones which started from prepubertal period and continue up to post menopausal period. Reproductive life span is determined by the histological changes of ovarian follicles. The study was carried out to counting the total number of follicles in relation to age.


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The ovaries are paired female reproductive organ which are situated in the lesser pelvis; one on each side of the uterus 1 .In the young adult, they are almond shape, solid and white in colour, approximately 3cm long, 1.5cm wide and about 1cm thick 2 .It undergoes various changes under the influence of hormones which starts from prepubertal period and continues up to post menopausal period 3 .
Follicles are the functional unit of the ovary.Most of the follicles (90%-95%) are primordial follicles.As the follicles increase in size, it passes through three developmental stages: primary follicles, secondary follicles and tertiary (mature or graafian) follicle.Those follicles which do not ovulate, degenerate by a process called atresia.Following ovulation of the large dominant follicle, the follicle wall collapse into folds and the units transforms into a corpus luteum.The corpus luteum ultimately degenerates becoming a white scar of dense connective tissue, the corpus albicans 4 .
Ovary fulfills two major objectives: generation of fertilized ovum and the preparation of endometrium for implantation through the sequential secretion of estrogen and progesterone hormones 5 .These activities are integrated into cyclic repeatitive process of follicular maturation, ovulation with formation and regression of corpus luteum under the control of the hypothalamo-hypophyseal system 6 .
Regrettably however, the ovaries as organs that become completely depleted in menopause.In fact, this depletion of the follicular population of the ovaries begins very early even in embryonic life, continues throughout childhood, increases at each ovarian cycle along the menarche and is finished at menopause 7 .Besides these, ovary possesses many pathological disorders including cyst, inflammation, carcinoma.There are a great variety of hormonal disorder, most commonly due to endocrine imbalances, complications of pregnancy and neoplastic proliferation 8 .Considering the enormous anatomical and physiological overload, the reproductive lifespan is ultimately determined by the number of follicles.By applying the accurate and efficient methods for estimating the number follicles are paramount for ovarian aging process 9 .The study of the follicular numbers can provide also important information about the function of the ovary, particularly in the relationship between folliculogenesis and the factors that regulate it 10 .

Procedure for the histological study
For histological study, formalin fixed right sided ovaries were taken.Six fresh samples were selected from each group.With the help of a sharp BP blade, ovary was opened by a median vertical cut extending from the one pole to another pole of the ovary and then another cut which sectioned the ovary transversely at its middle (Fig. 1a).A 5mm 3 rectangular enblock was taken from each part of the ovary for preparation of tissue block (Fig. 1b).Thus four pieces of blocks were taken for subsequent processing.
Four slides were prepared from each sample.Thus a total of twenty four (6×4=24) histological slides were made from each group.Finally, ninety six (24×4=96) slides were prepared for the whole study.

RESULTS
In

DISCUSSION
In the present study, the highest mean (±SD) number of the follicles were established in prepubertal age (group A) and the lowest were in postmenopausal age (group D).
According to Standring 2 40,000 ovarian follicles are present at puberty.Ryan, Berwokitz and Barbieri stated that by the first menses, the number of follicles is approximately 200,000.By 52 years of age, most women have loss all functional ovarian follicles and menopause has begun, menopausal ovary becomes devoid of functional follicles.Gartner &Hiatt 13 described that near the end of the 5 th fetal month, ovary contains about 5 to 7 millions oogonia.About 1 million of the oogonia become surrounded by follicular cells and survive to the time of birth.Of the 1 million oogonia that survive to become primordial follicles, 600000 become about 400000 follicles.
Faddy and Gosden 14 found that according to simple bi-exponential of ageing, 100000 follicles remain in the great majority in 21.

CONCLUSION
In the present study, it was observed that significant change of the total number follicles of the ovary occur with advancing age.
Tissue blocks were refixed in Carnoy's fluid in a plastic container.The tissues were washed in running tap water.Then dehydration was done with ascending grades of alcohol, cleared with xylene, infiltrated and embedded in paraffin.Paraffin blocks were cut at 5µm thickness and were stained with Mallory-Heidenhains Aniline blue (Mallory-Azan) stain.

Fig. 1
Fig. 1 Diagrammatic representation showing the cutting plane (a) & enblock of ovary for histological study

Fig. 2
Fig.2 representation of the use of counting circle in Diagrammatic (a) and Photomicrograph (b) counting the number of the follicles.Standardization for counting number of follicles per unit area of the microscopic field:Inner diameter of the counting circle corresponded with 52 divisions of the stage micrometer under 10x objective and 10x eyepieces.

Fig. 3
Fig. 3 Mean (±SD) total number of follicles in different age groups

Larsen 5
reported that by the onset of puberty approximately 30000 ovarian follicles are present.Ultimately 50 years later, this is finally exhausted, no follicles are present.

11 Study group distribution in different age groups Study groups Age range (in years) No. of samples (n = 65x2=130)
DMC),Dhaka.Approximate age & sex were noted down from the morgues record book at the time of collection of samples.Then the samples were brought to the Department of Anatomy, Sir Salimullah Medical College.Samples were distributed into four groups the present ovary, the mean (±SD) total number of the follicles in the microscopic field were 82.67  2.07 per square millimeter in group A, 43.33  8.17 per square millimeter in group B, 10.00± 5.47 per square millimeter in group C and 1.17  1.94 per square millimeter in group D. The differences of the number of the follicles were significant (P<0.01) between group C & D and highly significant (P<0.001) between the other age groups.The results are shown in Table1andFig 3.
15te15mentioned that by the fourth month of prenatal life, the ovaries may contain 5 million oogonia.By the time of birth, many of the oogonia have degerated and about 2 million of them are present.From birth to puberty, the number of primordial follicles declines to around 300,000-400,000; of these only about 400 continue oogenesis and are released from the ovary.The mean (±SD) values of the present study in reproductive age agreed with Standring, Larsen, Gartner & Hiatt and Faddy & Gosden .However, in prepubertal age, the values were lower than that of the findings of Ryan, Berwokitz & Barbieri and Seeley, Stephens & Tate.This discrepancy might be due to the differences in the methods of collection, fixation, staining and magnification.