The document discusses preclinical screening methods for anti-fertility agents. It describes various mechanisms by which contraceptives can prevent fertility, including inhibition of ovulation, prevention of fertilization, and interference with embryo development. Classification of contraceptives and in vivo and in vitro screening methods for evaluating anti-ovulatory, estrogenic, androgenic, and anti-androgenic activity are outlined. Key assays involve evaluating effects on ovulation and sexual organ weights in rodents, as well as receptor binding studies. The goal of preclinical screening is to identify potential anti-fertility compounds and understand their mechanisms of action before testing in humans.

1. PRECLINICAL SCREENING OF
ANTI FERTILITY AGENTS :
PRESENTED BY:
NAVEEN K L
Dept. Of Pharmacology
Srinivas College Of Pharmacy
Valachil , Mangalore
1

2. Contents :
• Introduction
• Mechanism of Anti-Fertility
• Classification of Contraceptives
• Screening methods for anti fertility drugs
• Reference
2

3. Introduction :
• Anti- Fertility agents are the agents, which prevents the fertility by
interfering with various normal reproductive mechanism, in both males
and females.
• Anti-Fertility agents are also called as Contraceptives agents. Basic aim
of anti-fertility drug is to prevent conception or fertilization.
• Oral contraceptives….. Steroids…… prevent menstrual cycle or
ovulation in females.
3

4. • Gonads (ovary & testis) secretes various hormones called gonadal
hormones
• Estrogen, Progesterone & Androgens.
• Main Functions:
*Development of primary or secondary sexual characteristic
* Reproduction
4

5. Hypothalamic -Pituitary-Gonadal axis:
Hypothalamus
Anterior
Pituitary
Gonads
(Ovary/Testes)
GnRH
LH FSH
+
+ +
Target
cells
Oestrogen, Progesterone & Testosterone
Inhibin
E/P/T
E/P/T LH/FSH
-
-
--
5

6. Menstrual
Flow
Proliferative Phase Secretory Phase
6

7. 7

8. Stages of the estrus cycle in rats:
• The estrus cycle is a cascade of hormonal and behavioral
events, which are highly synchronized and repetitive.
• The short and precise estrus cycle of the laboratory rats has
been a useful model for reproductive studies. The
laboratory rat is a spontaneous ovulating, nonseasonal,
polyestrus animal. It ovulates every 4-5 days throughout the
year unless interrupted by pregnancy or pseudopregnancy.
• The estrus cycle of a rat is usually completed in four to five
days.
• Rats and mice are spontaneous ovulators i.e. they do not
need the presence of male to induce the ovulation.
• Difference from Human Menstrual Cycle … The peaks of
oestrogen and progesterone are typically separated in
human but in rodent not i.e. overlapping.
8

9. Estrus:
In this stage the uterus is enlarged and extended due to fluid accumulation,
estrogen secretion is at its peak. In estrus stage the smear shows presence of
squamous cornified cells (hexagonal or pentagonal cells). Estrus means
period of heat and is characterized as a period of sexual receptivity, when the
female allows copulation. During this stage there is increased running activity.
This stage lasts for 12 hours.
Proestrus:
It is the beginning of new cycle. The follicles of the ovary start to mature
under the influence of gonadotropic hormones and estrogen secretion start
increasing; the smear is characterized by nucleated epithelial cells, the stage
last for about 12 hours.
9

10. Metestrus:
The ovary contains corpora lutea, secreting progesterone. This stage is
indicated by the presence of a mixture of cornified epithelial cells and
leukocytes indicating the postovulatory stage and desquamation of the
epithelial cells. Metestrus stage lasts for about 21 hours.
Diestrus:
The corpus lutea regress and the declining secretion of estrogen and
progesterone causes regression of the uterus. The smear shows only
leukocytes. This stage is the longest phase of the estrus cycle and has
duration of about 57 hours (Figs 8.2 to 8.7).
10

11. Mechanism of Anti-Fertility….
Inhibition of ovulation
Prevention of fertilization
Interference with
ovum transport
Destruction &
resorption of
early embryos
Prevention of
implantation of
fertilized ovum
11

12. Classification of Contraceptives:
Hormonal
Contraceptives
Oral pills
Depot
Preparation
Combined pills
Progesterone
only pills
Once a Month
pills
Male pill
Injectables
Subdermal
implants
Vaginal rings
12

13. Methods for females :
• In Vivo Methods
 Anti-ovulatory Activity :
• HCG-induced Ovulation in Rats
• Cupric Acetate-induced Ovulation in
Rabbits
 Estrogenic Activity :
• Vaginal Opening
• Assay for Water Uptake
• In Vitro Methods
• Estrogenic Receptor-binding Assay
Methods for males:
• In Vivo Methods
• Cohabitation Test
• Fertility Test
• Subsidiary Test
 Androgenic Activity
• In Vivo Methods
• Chicken Comb Method
• Weight of Ventral Prostate, Seminal
Vesicles and Musculus Levator Ani
• Nitrogen Retention
 Anti-androgenic Activity
• In Vivo Methods
• Chicken Comb Method
• Antagonisim of Effect of Testosterone on
Weight of Ventral Prostate, Seminal
Vesicles and Musculus Levator Ani
13

14. Antiovulatory Activity:
 HCG-induced Ovulation in Rats.
Purpose and Rationale
Method used for the screening of anti-ovulatory agents
Immature female albino rats do not ovulate spontaneously and do not show cyclic changes of
vaginal epithelium
Priming with human chorionic gonadotropin (HCG) induces follicular maturation, followed by
spontaneous ovulation 2 days later
Injection of anti-ovulatory drugs, prior to the induction procedure will prevent ovulation
14

15. Procedure:
Immature female albino rats 24-26 days of age are used for the experiment
The animals are treated with various test drugs in a different dose levels
After administration of test drug, HCG is given exogenously for ovulation
After 2 days, animals are sacrificed; ovaries are dissected out, preserved in 10%
formalin and subjected to histopathological evaluation
The results are compared with the control group.
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16. • Purpose and Rationale
This assay is based on the principle that vaginal opening occurs in immature female albino mice and rats
by treating with estrogenic compounds.
The sign of complete vaginal opening is observed as a sign of estrogenic activity.
Procedure:
Immature female animals (18-days old mice, 21-days old rats ) are used for the study
The test and standard drugs are administered to the animals Intramuscularly in cotton seed oil
The time of complete vaginal opening can be observed as a sign of estrogenic activity
Estrogenic Activity
 Vaginal Opening.
16

17. Purpose and Rationale
Estrogenic receptor binding assay uses the principle of competitive binding of
labelled and unlabelled estrogen on the estrogen receptors
Estrogenic compounds displaces the labelled estrogen in a concentration
dependent manner from the estrogen receptor
In Vitro Methods:
Estrogenic Receptor-binding Assay.
17

18. Procedure:
Cytosol preparation:
Uteri from 18-days old female albino mice are removed and homogenized at 0°C in 1:50
(w/v) of Tris-sucrose buffer in a conical homogenizer
Human endometrium from menopausal women is frozen within 2h of hysterectomy and
stored in liquid nitrogen
The frozen endometrium is pulverized and homogenized in 1:5 (w/v) of Tris-sucrose
buffer
Homogenates are centrifuged for 1h , determination of specific binding in mouse uterus
cytosol as a function of steroid concentration incubation time and temperature
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19. Triplicate aliquots of 125mL of cytosol are incubated with 5 or 25 nm labeled
steroid either for 2 or 24 h at 0°C or for 2 or 25 h at 25°C in the absence (total
binding ) or presence (non-specific binding) of a 100 fold excess of radio inert
steroid
Bound steroid is measured by Dextran Coated Charcoal (DDC) adsorption
19

20. Dextran coated Charcoal (DDC) adsorption Technique
A 100 µl aliquot of incubated cytosol is stirred for 10 minutes at 0°C in a micro titer
plate with 100 μl of DCC suspension (0.625% dextran 80,000, 1.25% charcoal Norit A)
and then centrifuged for 10 minutes at 800 g
The concentration of bound steroid is determined by measuring the radioactivity in a
100 μl Aliquot of supernatant.
For calculation of relative binding affinity, the percentage of radioligand bound in the
presence of competitor compared to that bound in its absence is plotted against the
concentration of unlabelled competing steroid.
20

21. Methods for the males :
 Cohabitation Test.
• Purpose and Rationale
This test determines the time interval for litter production after placing treated males
with 2 females.
The date of mating is calculated from the date of parturition. This method is suitable for
drugs known to cause several weeks sterility
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22. Procedure:
Adult female and male albino rats of proven fertility are used for the study
They are kept for mating in the ratio of 2:1 till both females deliver the litters
The date of mating is calculated from the date of parturition
The time interval for litter production after placing treated males with two
females is calculated
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23.  Fertility Test.
Fertility test is based on the evaluation of average litter size. Anti-fertility agents
negatively affect the average litter size.
Purpose and Rationale
Procedure:
Groups of 5-10 male rats of proven fertility are treated with drug and are
paired with fertile females in the ratio of 1:3 Daily vaginal smears are
examined for the presence of sperms
All females passed through 1 estrus cycle must have mated
The mated animals are kept separately till the completion of the gestational
period
23

24. The litters are counted and using the following formula average litter size is calculated
Total no. of litters
Average litter size = ———————————
No. of females mated
If vaginal smear shows leukocytes for 10-14 days, Pseudopregnancy is confirmed.
Fertility patterns can be obtained from changes in average litter size
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25.  Subsidiary Test:
Procedure:
Adult male albino rats weighing between 150-250 g are used for the study
They are kept in a cage containing artificial or animal vagina
Artificial vagina is the cylindrical plastic jacket with the rubber liner, filled with water at
5°C. 0.5 ml of ejaculate is diluted with saline containing traces of formalin
Resulting suspension counted on haemocytometer.
Purpose and Rationale
This test determines the changes in spermatozoa count with time. The anti-fertility
drugs affect the spermatozoa count negatively
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26. Androgenic Activity:
This assay is based on the principle of androgens affect the secondary sex organs in
male individual
In the rats, the growth of the ventral prostate, the seminal vesicle and the
musculus levator ani depend on the presence of male sexual hormones
Weight of Ventral Prostate, Seminal Vesicles and Musculus Levator Ani
Purpose and Rationale
26

27. Procedure:
Immature male rats weighing about 55g are orchidectomized
The animals are treated with the test compounds in various doses orally in 0.5 ml 0.5% carboxymethyl
cellulose or 0.2 ml sesame oil suspension daily over a period of 10 days
Testosterone given subcutaneously in doses of 0.02, 0.1 and 0.5 mg per animal, or methyl testosterone in
doses of 0.25, 1.5, and 5 mg per animal, serve as standard
Controls receive the vehicle only. On the eleventh day, the animals are sacrificed and the seminal vesicles, the
ventral prostate, and the musculus levator ani carefully dissected and weighed
The weight of control and test group is compared with suitable statistical analysis
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28. Anti-androgenic Activity:
In the rats, the growth of the ventral prostate, the seminal vesicle and the musculus
levator ani stimulated by testosterone, anti-androgenic compound inhibits this effect.
Antagonisim of Effect of Testosterone on Weight of Ventral Prostate, Seminal Vesicles
and Musculus Levator Ani.
Purpose and Rationale
28

29. Procedure:
Male rats weighing 50-70 g are castrated and one day after surgery, the rats are
injected once daily for 7 days with 0.15 mg testosterone propionate in 0.1ml
sesame oil
The test compound also dissolved or suspended in sesame oil at various doses and
injected subcutaneously daily at a separate site for 7 days
Controls receive testosterone injections only. On 8th day, the animals are sacrificed
and weights of ventral prostate, seminal vesicles and musculus levator ani weighed
The weight of control and test group is compared with suitable statistical analysis
29

30. Reference:
• Sciarra JJ. The continuing need for contraceptive research. Fertil Steril
1981;36:697-8.
• Kostyk SK, Dropcho EJ, Moltz H, Swartwout JR. Ovulation in immature rats in
relation to the time and dose of injected human chorionic gonadotropin or
pregnant mare serum gonadotrophin. Biol Reprod 1978; 19: 1102-7.
• Turner RA. Screening methods in pharmacology. New York: Academic Press
1971:85-118.
• Vogel HG, Vogel WH, Editors. Drugs Discovery and Evaluation. Berlin: Springer
1997.
• Ghosh R. Modern concept on pharmacology and therapeutics. (24th ed).
Calcutta, Hilton and Co, 1991.
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