CONTRACEPTION

CLINICAL PROFILE AND TOXICOLOGY STUDIES ON FOUR WOMEN IMMUNIZED WITH Pr-li -HCG-TT

S.

Kumar,

N.C.

Sharma, J.S. Bajaj, and V. Hingorani

G.I’.

‘I‘alwar

Departments of Obstetrics and Gynaecology, Medicine and Biochemistry. All India lnstitute of Medical Sciences, New Delhi-110016, India

ABSTRACT Four women in child bearing age group were immunized with the vaccine Pr-B-HCG-TT. All of them responded to active isoimmunization by production of anti-HCG and anti-TT antibodies. All subjects were followed up for one year. A thorough clinical examination was done at monthly visits and nothing abnormal was detected. Laboratory examination included (i) Hepatic function (serum bilirubin,.alkaline phosphatase, serum transaminases GOT and GPT, LDH isoenzymes, cholinesterase), (ii) Renal function tests (urine routine and microscopic examination, blood urea and serum creatinine and urinary creatinine, (iii) Metabolic studies (blood glucose. serum proteins, ’ serum cholesterol, and free fatty”acids); (iv) Endocrinal study (protein-bound-iodine, insulin tolerance test, serum progesterone and endometrial biopsy) and (v) Haematological investigations (haemoglobin, total and differential leucocyte count, erythrocyte sedimentation rate, platelet, reticulocyte count and peripheral smear). No abnormality of any kind has been noticed so far, thus indicating that active isoimmunization with Pr-A-HCG-TT has no adverse or undesirable side effects.

Request Head of Accepted

for the for

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reprints to be sent to Department of Obstetrics publication

October

1976 VOL. 13 NO. 2

Professor V. Hingorani, and Gynaecology.

20,

1975

253

CONTRACEP

INTRODUCTION Regulation of human fertility by active immunization with Pr-B-HCG-TT is a novel approach. If oroved safe and effective. ’ this method offers obvioui advantagesAover other methods of contraception, specially in developing countries with limited resources. In a preliminary study in four women volunteers, Pr- B- HCGTT was found to be immunogenic and all subjects responded by production of anti-HCG and anti-TT antibodies with a lag period of J-6 weeks (1). These subjects attended the clinic at monthly intervals and underwent a complete clinical examination. Their haematological, endocrinal, metabolic, hepatic and renal function tests were done to assess adverse effects, if any, attributable to immunization. The clinical and laboratory data is being presented in this communication. MATERIALS AND METHODS Selection of Subjects: Healthy multiparous women in child _p bearing age group, who hZ?f-completed their families and had undergone bilateral tubal ligation, participated in this study. The clinical profile of these subjects is given in Table I. These subjects were interviewed every month and general physical, systemic and pelvic examination was carried out at every visit. A record of body weight and menstrual data was maintained. The following investigations were Toxicology Studies: done at variable intervals, after the demonstration of antiHaemoglobin and packed cell volume were done HCG antibodies. as described by Dacie and Lewis (Z), serum bilirubin and creatinine by methods described (3), alkaline phosphatase, serum and urinary proteins as described by Wootton^ (4), cholinesterases as described bv Iobal and Talwar (5). total LDH was estimated by method of’Wri?blewski and LaDue’i6), SGOT and SGPT estimated by calorimetric method of Reitman and Frankel (7), LDH isoenzymes by disc-electrophoresis (8), blood urea and true blood glucose estimation by autoanalyzer cholesterol by method of Chiamory and Henry (lo), free (9)) fatty acids were estimated by calorimetric ultramicromethod of No&k (ll), cortisol was estimated by fluorimetric method of Mattingly (12), insulin tolerance test as advocated by serum progesterone by radioHall, Anderson and Smart (13), immunoassay (14) and protein bound iodine as described by Barker et-- al. (15). RESULTS

tion,

254

Throughout the period of Clinical Examination: no znormality was detected_ in general physical,

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observasystemic

1976 VOL. 33 NO. 2

K.W.

A .M .

N.D.

B.H.

1.

2.

3.

4.

Name

_.__~

26

30

32

30

Age yrs

P4+1

P3+2

P2+1

P4+1

T: CLINICAL

I,

Full term normal delivery

II

Medical termination of pregnancy

-________

Termination of last pregnancy (TLPI

--. _-._.~- ..----

Parity

TABLE

__

(1

Post-partum ligation

II

Hysterotomy and ligation

_~-__._

Operative procedure

DATA

9 Days

9 Days

23 Days

Interval between TLP and administration of Pr-B-HCG-TT A

.@

ti

*

Normal

for seven months.

6-8

amenorrhea

26-28

Lactational

I j K . II’ .

MENSTRUAL

5-6 4-5 5-6 2-3

24-27 30-43 26-30

Duration

Post-immunization menstrual history

DATA

22-28

Cycle

__. -_.____-_~_~~-~--

IT:

Pre-immunization menstrual histor) ~---~--.__----___----Amount Duration Cycle

,~__-____~.__________.__~__.--_~

Name

TABLE

11

,t

It

Normal

Amount

-

-

8

12

12

12

(Months)

Period of follow-up

I

5 9 10

9

1 8

A.M.

N.D.

B.H.

Traces 15.85

35.54

20.86

34.60 26.95

Level of anti-HCG antibodies*

15.27

6.0

6.4 7.8

9.58 7.85

cycle of 43 days following

lactational

of serum

Secretory

Proliferative

?I ,,

Secretory ,t

Endometrial biopsy

23 27

27**

27 23 23

20 21

Day of menstrual cycle

HISTOLOGY

amenorrhea.

LEVELS AND ENDOMETRIAL

Serum progesterone ng/ml

SERUM PROGESTERONE

bound at 1:lO initial dilution

III:

** Second menstrual

% "'1-HCG

5 10

K.W.

*

No, of treatment cycle

Name

TABLE

,

CONTRACEPTION

and pelvic examination. There normal limits. weight.

Blood pressure was no significant

remained within change in body

Menstrual Data and Indices of Ovulation are given in Tables II and IIT.Serum~proges-~~~~~~-~~~ between 6.0 to 15.27 ng/ml in the four subjects. These values being over 3 ng/ml indicate ovulatory cycles (16). The endometrial biopsy studies indicated secretory endometrium in 3 out of 4 subjects (Figures 1, 7, 31.

Figure

258

1:

Endometrial biopsy in premenstrual (K.W. early secretory endometrium

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phase showing - cycle 5).

1976 VOL. 13 NO. 2

CONTRACEPTION

Figure

2:

Endometrial biopsy in premenstrual secretory endometrium (K.W. - cycle

phase 10).

showing

Figure

3:

Endometrial biopsy in premenstrual secretory endometrium (A.M. - cycle

phase 10).

showing

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1976

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13 NO. 2

259

CONTRACEPTION

The exception was N.D. where the be accounted for irregular cycle amenorrhea (Figure 4, Table II).

proliferative phase could length following lactational

Figure

second cycle following showing proliferative

4:

Endometrial lactational endometrium

biopsy in amenorrhea (N.D.).

The same Table also indicates following immunization in the

unaltered menstrual other 3 subjects.

cycles

Liver Function: Detailed assessment of hepatic function No dewas carr‘iE;d?%m--results are shown in Table IV. SGOT and SGPT values monstrable adverse reaction was noted. in four subjects showed no deviation from normal, reflecting normal hepatocellular function. Results of metabolic tests are given Metabolic Tests: Serum protein profile is within the range of in Table V. as are blood glucose, serum cholesterol normal observation, and free fatty acids. Results of Renal Function Tests: p renal function test are given in Table deviation from normal. are to

260

Haematological shown in Table Insulin evaluate

Results Data: These are VII.

investigations for These show no VI.

of haematological within the normal

tolerance test was done in one hypothalamic-hypophyseal-adrenal

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studies range.

subject (N.D.) axis (Figure

1976

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5).

13 NO. 2

CONTRACEPTION

-

20

d

rn

-

30

0

60

90

10

?I % 3 u

0

TIME ( MINUTES ) Figure

5:

Insulin

tolerance

test

(N.D.).

This test showed a normally functioning hypothalamic-hypoas the rise in plasma cortisol was 12.5 physeal-adrenal axis, value obtained during the test was 30.0 ug% and the highest ug%. All the subjects did not show any increase in the neck Protein bound circumference and were clinically euthyroid. iodine varied from 5 - 7.5 ngm/lOO ml, normal range being 5 - 8 ngm/lOO ml. DISCUSSION A careful clinical examination of these immunized subThere was no signifijects did not reveal any abnormality. In three subjects (K.W., A.M. cant change in body weight. menstruation was regular and cycles were ovulatory, and B.H.), as confirmed by serum progesterone levels and endometrial menstruation was irregular biopsy. In one subject (N.D.), following lactational amenorrhea, though there was evidence of ovulation as serum progesterone level was 6 ng/ml in prePresence of ovulation menstrual phase in one of the cycles. indicates no untoward effect of immunization on hypothalamicpituitary-ovarian axis. Results of liver function tests are within normal range in all subjects, reflecting normal hepato-cellular function. Renal function tests as evaluated by urine examination, blood urea and serum and urinary creatinine show no impairment.

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1976 VOL. 13 NO. 2

26X

3-13KAU/lOOml

(Pseudo)

Acetyl-

25-40 AA/min/ml

15-30 AA/min/ml

-

-

36

24

11

7

2-15 I.U./l

SGPT

Cholinesterase, (true)

15

17

6.5

0.6

Z-20 I.U./l

6.5

0.7

K.W.

SGOT

phosphatase

Alkaline

range

LIVER FUNCTION

0.2-0.8 mg/lOO ml

Normal

IV A:

Serum bilirubin

Tests

TABLE

0.3

35

30

9

17

-

-

4

8

12.5 9

0.3

A.M.

Observed

TESTS

28

15

17

16

16

0.7

-

-

21

9.5

9.5

0.3

N.D.

values

-

13

31

7.5

34

20

5

3

13

0.5

B.H. 0.3

-

I

* Only 8 subjects

LDH Iso-enzymes

I II III IV

IV B:

Lactic dehydrogenase

Tests

TABLE

I.U./l

SO-170

investigated.

15-30* 20-40 15-35 10-35

range

Normal

LACTIC DEHYDROGENASE

A> 144 21.4 25.3 25.7 27.6

K.W. 168 26.3 25.0 20.8 28.0

Observed

PATTERNS

120

168 20 28.3 26.3 25.3

B.H _-L N&

values

(LDH) AND ITS ISOZYMIC

j

6.2,7.8

6-8g/lOOml

1.1-1.5

Serum protein

A.G. Ratio 1.x,1

6.2,7

900

93-750p.eq./l

Serum free fatty acids

1.5,-

242

200

150-250mg/lOOml

74

A.M.

Observations ---__

Serum cholesterol

K.W.

~~

INVESTIGATIONS

64

range

METABOLIC

60-100mg/100ml

Normal

V:

Random blood sugar

Tests

TABLE

1.2,1

6.5,7

775

206

68

N.D.

l.l,-

6.9,7.5

450

154

70

B.H.

CONTRACEPTION

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VOL. 13 NO. 2

265

0-ZOmm

ESR (Wintrobe)

Normocytic normochromic

150,000-300,000

Platelets

M Z-10",

L 20.45%

iPeripheral film

I

I

I

DATA

146,000

Normocytic hypochromic

1

18

11

E l-69,

B < 1%

70

P 40-759,

I

/DLC

1.6

24

6000

O.?-2%

fall 1st hr

4000-llOOO/ml

TLC

30

10

K.W.

HAEMATOLOGICAL

/Reticulocyte

I

35 to 47%

Packed cell volume

range

11.5-16.4g/lOOml

Normal

VII:

Haemoglobin

Tests

TABLE

200,000

Normocytic normochromic

0

38

2

60

1.0

18

7600

40

13

A.M.

138,000

Mild hypochromic

2

39

4

55

2.8

38

5000

33

10.8

N.D.

Observations

-~--

158,000

Normocytic hypochromic

2

34

6

58

2.5

20

6000

30

10

B.H.

CONTRACEPTION

The haematological data reveals normal haenatopoiesis. The cholesterol and free fatty serum protein, blood glucose, Protein bound iodine in all acids are within normal range. the subjects was within normal range indicating euthyroid The normal insulin tolerance test in one subject status. (N.D.) reflects normalcy of hypothalamic-hypophyseal-adrenal The period of follow-up ranged from eight months to axis. one year and the results of detailed investigative work-up demonstrate that immunization with Pr-B-HCG-TT does not proThis duce any adverse effects on relevant body systems. at present seems to be safe. However, a regime, therefore, further period of observation will be necessary to confirm the long-term safety of this approach. REFERENCES 1.

Talwar, G.P., Dubey, S.K., Salahuddin, M., Das, IIingorani, Antibody response V. and Kumar, S.: Contraception HCG-TT vaccine in human subjects. 13: 737 (1976).

2.

Dacie, J.V. and Lewis, J. 2 A. Churchill Ltd.,

3.

Henry, niques.

3.

Wootton, .J. 4 A.

5.

Iqbal, 2. and Talwar, G.P.: Acetyl cholincsterase in developing chick embryo brain. .J . Neurochem. 18: 1261. 1267 (1971).

6.

Wrbblewski, F. and LaDue, activity in blood. Proc. 210-213 (1955).

7.

Reitman, S. and Frankel, S.A.: Calorimetric method for the determination of serum glutamic oxaloacetic and glutamic pyruvic transaminases. Amer. J. Clin. Path. 28:56-63 (1957).

8.

Davis, B.J.: Disc-Electrophoresis application to human serum proteins. Sci. 121:404-4:: (1964).

9.

Autoanalyser New York.

10.

S.M.: Practical London, 1968.

Haematology.

R.J.: Clinical Chemistry. Principles Harper and Row, New York, 1966. I.D.P.: Churchill

Micro-analysis Ltd., London,

Methodology

J.S.: Sot.

C., to Pr-B-

in Medical 1964.

and TechBiochemistry.

Lactic dehydrogenase Exp. Biol. N. Y . 90:

N. 16b.

- II.

Method Ann. N.Y.

Technicon

and Acad.

Corporation,

Chiamory, N. and Henry, R..J.: Study of the ferric chloride method for determination of total cholesterol and cholesterol esters. Amer. J. Clin. Path. 31: 305309 (1959).

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CONTRACEPTION

11.

Novak, M.: Calorimetric ultramicromethod for the determination of free fatty acids. J. Lipid Res. 6:431-433 (1965).

12.

Mattingly, D.: A simple fluorimetric method for the estimation of free 11-hydroxy corticoids in human plasma. J. Clin. Path. 15:347-379 (1962).

13.

Hall, R., Anderson, J. and Smart, G.A.: Fundamentals of Clinical Endocrinology. Pitman Medical Publishing Company, London, Second Edition, 1974.

14.

Laumas, K.R., Rahman, S.A., Hingorani, V., Puri, C.P., Baliga, N. and Laumas, V.: Hormonal changes in patients undergoing therapeutic abortion with prostaglandin FZu, 15(S) 15-methyl prostaglandin F2d, and hypertonic

268

saline.

Contraception

10:617-636

(1974).

15.

Barker, S.B., Humphrey, M.J. and Soley, M.H.: Clinical Determination of Protein Bound Iodine: J. Clin. Invest. 30:55 (1951).

16.

Israel, R., Mishell, D.R., Jr., Stone, G.G., Thorneycroft, I.H. and Moyer, D.L.: Single luteal phase serum progesterone assay as an indication of ovulation. Am. J. Obstet. 6 Gynec. 112:1043-1046 (1972).

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