ANTIFERTILITY

1. Dr. Prerna Singh
Junior resident
Department of Pharmacology
JNMC, AMU

2.  Prevent fertility by interfering with reproductive mechanism
 Ideal contraceptive:
 100 % effective
 Safe
 Easy to use
 Reversible
 Affordable
 Appropriate to use at all stages of reproduction

3. Inhibition of ovulation
Prevent fertilization
Interfere with ova transport
Interfere with implantation
Distraction of early implanted embryo

4. Female
fertility
Anti
ovulatory
activity
HCG
induced
ovulation
Cupric
acetate
induced
ovulation
Estogenic
methods
In vivo
1.1.Vaginal
opening
2.Assay for
water uptake
3.Vaginal
cornification
4.Chick oviduct
method
In vitro
•1.Estrogenic
receptor
binding assay
•2.Potency
assay
Progestational
activity
In vivo
1.Pregnancy
maintenance
test
2.Proliferatio
n of uterine
endometrium
3.Carbonic
anhydrase
activity
4.Deciduoma
reaction
In vitro
Progesterone
receptor
binding assay

5.  Rationale: HCG induces follicular maturation and spontaneous
ovulation after 2 days
 Procedure: Immature female albino rats(24- 26 day)
 Immature rats do not ovulate spontaneously and do not show cyclical
change in vaginal epithelium

6. Albino rats: test drug in various dose
HCG
2 days later: rats sacrificed and ovaries
dissected.
Histopathology compared with control

7.  Rabbit - reflex ovulator
Ovulate few hours after mating
 mechanical stimulation of vagina
Presence of male
Female albino rabbit – 3-4 kg

8. Isolate rabbit for 21 days
Test drug
24 Hr later
1%Cupric acetate 0.3 ml/kg IV in
0.9% NS
Sacrifice after 24 hr
Number of ovulation points recorded
Histopathology of ovaries and uterus

10. Estrogen in immature albino rats(21 day old) – vaginal
opening occurs
Test and standard given IM in
cotton seed oil
Time of complete vaginal
opening observed

11. Estrogen- increase uptake and retention of water
Peak at 6 hr
Ovariectomized rat used
Test compound given in cotton seed oil SC
Sacrifice 5 hr after treatment
Excise uterus; weight; dry weight after drying
in oven at 100 degree for 24 hr; compare with
control

12. Estrus cycle: cascade of hormonal behavioral event and
highly synchronized and repetitive
Estrogenic drugs change animal to estrus stage

13. • Corpus lutea
• Progesterone
• Smear: leucocyte,
cornified cell
• 21 hr
• Corpus lutea regress
• Estrogen progesterone
regress
• Smear: leucocytes
• 57hr
• Uterus enlarged
extended – water
accumulation
• Estrogen peak
• Smear: squamous
cornified cell
• 12 hr
• Beginning
• Follicle start maturing
• Smear: nucleated epithelial
cell
• 12 hr
Pro-estrus Estrus
Met estrusDiestrus

15. Rats with regular estrus cycle are ovariectomized
Test and standard drug given In cotton seed oil oral
or SC for 4 days
Vaginal smear examined for cornified cell, leucocytes,
epithelial cell
Twice a day for 4 days
MODIFICATION: direct application of drug to vagina

16. 7 day old chick
Inject test twice daily for 6 days SC
Sacrifice and weight of body and oviduct
compare ratio of oviduct weight to body weight of
control(0.02mcg- 0.5 mcg estradiol)
Estrogen - proliferation of oviduct in chicks

17. Estrogen along with antiestrogen given
Estrogen dose is that which is required to produce 50% of
maximum response
Water uptake, utero-trophy, cornification seen
In vitro:
Aromatase inhibition: Compound which inhibits aromatase
(estrogen synthase) possess anti- estorgenic activity

19. Ovariectomy done on 8th day of pregnancy
Standard and test drug are given for 13 days
along with estradiol
Rats killed on day 21
Average of Living fetus at end of experiment
counted

20. Prime with estradiol(0.5mcg/ml) daily
for 7 days
Test drug for 5 days
Sacrifice after 24 hours
Uteri dissected, histology done

21.  Linear relation between progesterone and carbonic anhydrase activity
Immature female albino rats
Primed with estradiol
Test and standard drug given
Sacrifice, uterus remove,
Evaluate carbonic anhydrase activity in
endometrial extract by calorimetry

22.  Principle: tumor formation by progesterone in ovariectomized rats
Ovariectomized rat primed with 1 mcg estradiol for 4
days
Test drug for 9 days
Day 5 – 1mg of histamine dihydrochloride injection in
lumen of uterine horn
Sacrifice
Horn cut off
Weight and histology compared

23. IV Oxytocin on day 30 of pregnancy – abortion
Prior progesterone – prevent it
Test drug
24 hr later 10U oxytocin IV day 30
Prevent abortion
Control will abort in 2- 30 min

24. PRINCIPLE: Competitive binding of labeled & unlabeled
progesterone on progesterone receptors.
 Progesterone competitively displace labeled progesterone in
a concentration dependent manner from progesterone
receptor

25. Albino rats in pre estrous or estrous phase
mated
Male: female= 1:3
Examine vaginal smear every morning for
spermatozoa
Day1 pregnancy – when sign of mating seen;
separate them then
Test drug
Day 10 of pregnancy- laparotomy done,
implants in uterus and uterine horn counted
number of corpus luteum
Complete pregnancy : count litter if any

26.  Pre implantation loss: number of CL on D10 – number of implantation on D10
 Post implantation loss: number of implant on D10 – number of litters delivered
 % Implantation loss:
𝑛𝑜.𝑜𝑓 𝐶𝐿 −𝑛𝑜.𝑜𝑓 𝑖𝑚𝑝𝑙𝑎𝑛𝑡
𝑁𝑜.𝑜𝑓 𝐶𝐿
× 100
 %Post implantation loss
𝑛𝑜.𝑜𝑓 𝑖𝑚𝑝𝑙𝑎𝑛𝑡 −𝑛𝑜.𝑜𝑓 𝑙𝑖𝑡𝑡𝑒𝑟𝑠
𝑛𝑜.𝑜𝑓 𝑖𝑚𝑝𝑙𝑎𝑛𝑡
× 100
 % Antifertility activity
𝑛𝑜.𝑜𝑓 𝐶𝐿 −𝑛𝑜 𝑜𝑓 𝑙𝑖𝑡𝑡𝑒𝑟𝑠
𝑛𝑜.𝑜𝑓 𝐶𝐿
× 100

27. Confirm pregnancy by palpation after day 12
Intra amniotic and intra placental injection
under anesthesia on day 20
Effect: vaginal bleed
Weight change
Palpation

28. Interference with spermatogenesis without loss of libido &
secondary sexual characteristics
In vivo
1.Cohabitation
2.Fertility test
3.Subsidiary test
In vitro
1.Spermicidal activity
2.Immobilization assay
3. Non specific
aggregation estimation

29. Male female in 2:1 ratio kept for mating
Till both female deliver
Date of mating – from date of parturition
Time interval for litter production after placing
treated males with 2 females is calculated

30.  Anti-fertility agents negatively affect average litter size
Male female paired in 1:3 ratio
Vaginal smear daily – for presence of
sperm
Mated animal kept separately
Average litter size =
𝑡𝑜𝑡𝑎𝑙 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑙𝑖𝑡𝑡𝑒𝑟
𝑛𝑜.𝑜𝑓 𝑓𝑒𝑚𝑎𝑙𝑒𝑠 𝑚𝑎𝑡𝑒𝑑

31. Determine changes in spermatozoa count with drug
Rats kept in cage with artificial or
animal vagina
Ejaculates diluted with saline
containing traces of formalin
Suspension counted on
hemocytometer

32. Spermicidal drugs diluted in saline & serial
dilkution made in 0.2 ml of human seminal fluid
with 1ml of spermicidal sol.
Mixture incubated at 37oC for 30 min
A drop placed on slide & at least 5 fields were
observed under 400X for assessment of sperm
morphological change & motility

33. Human sample from 10 normal subjects after 72-96
hrs. of sexual abstinence subjected to routine semen
analysis count >100 million/ml & viability > 60% taken
for test
mixed in 1:1 ratio with different conc. of drugs
drop of mixture placed on a slide & at least 5 field
were microscopically observed under 400X for
assessment of motility.

34. Diff. conc. of drug mixed with ram sperm
suspension in1:1 ratio & kept at 37oC for 1 hr
Then from bottom of centrifuge tube, one
drop of sediment sperm placed on a slide &
% aggregation examined under 400X
the non-aggregated spermatozoa will remain
in supernatant, it collected & turbidity
determined spectrophotometrically at 545nm
Aggregation is indirectly proportional to sperm viability.

35.  1.CHICKEN COMB METHOD
PRINCIPLE: Growth of cap on comb by androgenic compounds
PROCEDURE:
 Sum of length plus height of each comb determined with mm ruler
 Capons inj. daily IM for 5 days with solution of test & standard drug in
1ml olive oil
 24 hrs after last inj. comb re-measured
 Compared statistically

36. PRINCIPLE: Androgen affect secondary sexual characters
PROCEDURE:
 Orchidectomy of Immature male rats of weight 50gm done
 Animals treated with test drug in different doses in 0.2 ml sesame oil
daily for 10 days
 In standard methyl testosterone given SC in 0.25, 1.5, 5 mg per
animals
 Control given vehicle only
 On 11th day animals scarified & seminal vesicles, ventral prostrate &
musculus levator ani dissected & weight & compared

37. . CHICKEN COMB METHOD
PRINCIPLE: Inhibition of growth of capon by anti-androgenic comp.
PROCEDURE:
 1-3 day old male white Leghorn chicks used
 Feed from day 1 on testosterone mixed in finely ground chick starting
mash at conc. of 80mg/kg food
 Test comp. dissolved in sesame oil & 0.1 ml inj sc daily for 4 day
 Control chicks receive only vehicle
 24 hr after last inj. Animals scarified & comb removed & weighed rapidly
& compare

38. 2. ANTAGONISM OF EFFECT OF TESTOSTERONE ON
WEIGHT OF VENTRAL PROSTARTE, SEMINAL
VESICLE & MUSCULUS LEVATOR ANI
PROCEDURE:
Same as for androgenic activity with addition of test drug
given simultaneously SC dissolved in sesame oil at a
separate site for 7 days.

39. S.K. Gupta Drug screening methods
H. Gerhard Vogel (Ed.) Drug Discovery and
Evaluation