Therapeutic abortion with the use of prostaglandin F2α

Therapeutic abortion with the use of prostaglandin F,, A study of efficacy, levels with

tolerance,

intravenous

P.

G.

GILLETT,

R.

A.

H.

and plasma

administration M.D.,

KINCH,

M.B.,

C.M.,

M.R.C.O.G.,

F.R.C.S.(C)

B.S.,

F.R.C.O.G.,

F.R.C.S.(C),

F.A.C.O.G. L.

S.

C.

PACE-ASCIAK,

Montreal,

WOLFE,

Quebec,

M.D.,

PH.D. PH.D.

Canada

Recent reports indicate that certain jrostaglandinr are effective abortifacients through their oxytocic activity. In this study, 10 women, 10 to 15 weeks pregnant, received prostaglandin F@ (PGF,a) by intravenous infusion for termination of pregnancy. Complete abortion was achieved in 9 cases with a mean induction-abortion interval of 24 hours, 41 minutes and a mean drug dose of 93.5 mg. An infusion rate of 50 pg per minute was suficient to induce uterine activity adequate for abortion. The incidence of side effects at this dosage level was high. Electrocardiogram changes and pyrexia were more pronounced at higher infusion rates. Plasma progesterone levels fell only after abortion. Mean plasma PGFIa levels before infusion were 0.40 ng. per milliliter and increased significantly with increases in infusion rate to a mean level of 3.00 ng. per milliliter at 200 pg per minute. Mean plasma PGFza levels double the preinfusion levels appeared sufficient to initiate adequate uterine activity. We conclude that, although intravenous PGF# is an effective abortifacient, this method of delivery has limited clinical Usefulness.

animportantnew class of naturally occurring biologically active substances, are potent stimulants of uterine contractility in nonpregnant women as well as in early and late pregnancy.lm3 Prostaglan-

din E,, E,, and F?LY (PGF,a) given intravenously have been used successfully to induce labor at or near term4-6 and to induce abortion in the first and second trimesters7p ’ The results obtained by various groups using intravenous PGF2~ as an abortifacient show variable success rates but have given some indication of dose-response relationships9-I1 and of side effects which included nausea, vomiting, diarrhea, and tissue reaction at the site of injection. The present study was designed to obtain further information on the efficacy of and tolerance to intravenous PGF,(u for the induction of therapeutic abortion in the first and second trimesters. At various intervals during the inductions as well as before and after, plasma levels of

PROSTAGLANDINS,

From the Department of Obstetrics and Gynaecology, Montreal General Hospital, and the Donner Laboratory of Experimental Neurochemistry, Montreal Neurological Institute, McGill University. Presented by invitation at the Twenty-seventh Annual Meeting of the Society of Obstetricians and Gynaecologists of Canada, Halifax, Nova Scotta, Canada, ]une 14-l 7, 1971. This study was supported by the Upjohn Company and Grants MT-1345 and MA-4181 from the Medical Research Council of Canada. 330

Vnlume Number

Therapeutic

112 3

Table I. Administration

50

pg/c.c.

Infusion (c.c./min.)

rate

0.5 1.0 2.0 4.0

PGF2a! were determined by a specific mass spectrometric isotope dilution assay based on a procedure reported by Samuelsson and coworkers.12 Patients

and

with PGF,n

331

schedule of PGF,ar

Concentration

Drug PGFza

abortion

methods

Ten healthy women (8 Caucasians, 2 Negros) aged 17 to 29 years recommended for therapeutic abortion were studied. The gestational ages varied between 10 and 15 weeks. Eight patients were primigravidas, and two were para 2. Patients with a history of habitual abortion (2 or more), spontaneous abortion in the previous 9 months, or evidence of attempted, threatened, or inevitable abortion in the present pregnancy were excluded. Also excluded were patients with uterine anomalies, a history of incompetent cervix, uterine operation, active pelvic gynecologic disorders, anemia, cardiac, renal or hepatic disease, asthma, or glaucoma. Drug formulation and dosage schedule. PGF,a: (U-14,583) as the tris (hydroxy methy1) aminomethane salt was supplied by Dr. Edward L. Masson of the Upjohn Company, Toronto, Ontario. Seventy-five milligrams of PGF,ar were diluted with 1,500 ml. of 5 per cent glucose and water to give a final concentration of 50 pg per milliliter. This solution was administered intravenously by a Harvard Pump in stepwise increments for a 12 hour period according to a schedule proposed by the Upjohn Company in Protocol 002 (Table 1). The infusion schedule was maintained for the full period irrespective of cervical dilatation or abortion. The dosage level was reduced to the next lower infusion rate only when a nontolerated level was encountered. If complete abortion had not occurred during the initial infusion, the patient was

Dosage ffdmin.) 25 50 100

200

Time (hr.) 4% 4 3%

Amount (w.) 0.75 12.0 24.0 42.0

observed during the following 12 hours. At the end of this interval, if complete abortion had not occurred, reinfusion of the drug was commenced at a dosage level selected by the investigator. Reinfusion was continued until complete abortion, a maximum administration of 75 mg. of drug, or until a maximum reinfusion time of 12 hours. If reinfusion did not produce complete abortion, a further 12 hour observation interval was allowed, following which, if abortion had not occurred, the pregnancy was terminated by other means. Measurement of uterine activity. In 8 of the 10 patients, uterine contractility was monitored during initial infusion. A 16 gauge Tuohy needle was introduced into the amniotic sac transabdominally in the midline 2 to 5 cm. above the symphysis pubis. When free reflux of amniotic fluid was observed, an 18 gauge polyethylene epidural catheter (Portex) was inserted through the needle, connected to a Hewlett-Packard transducer (1280C) attached to a Hewlett-Packard recorder (8020A, 8025A). The transducer dome and catheter were filled with sterile distilled water containing heparin (10 +g per cubic centimeter). Spontaneous uterine activity was recorded for 15 minutes prior to infusion of PGF,cy. Other measurements. The patients remained under close observation throughout the study. Temperature, pulse, and respiratory rate were determined every half hour, and blood pressure was determined every quarter hour during infusion. After each infusion period, vital signs were measured every two hours for a further 12 hours. Electrocardiogram monitoring was continuously maintained, and complete electrocardiogram tracings were obtained at each dosage level and during any apparent abnormality on the cardiac monitor.

332

Gillett

et al.

Laboratory analyses. The following laboratory tests were performed before infusion, at maximum tolerance level, and 12 hours following infusion : hemoglobin, hematocrit, leukocyte count, differential and platelet count, complete urinalysis, bilirubin, serum glutamic oxalacetic transaminase, serum glutamic pyruvic transaminase, alkaline phosphatase, serum creatinine, blood urea nitrogen, serum electrolytes, glucose, and progesterone. Measurement of prostaglandin F,CX concentration in plasma. Plasma concentrations were determined by a mass spectrometric isotope-dilution assay with the use of PGF,(u methyl (d3) ester as carrier. Twenty milliliters of blood was withdrawn before, at 2 hour intervals during, and 5 minutes after induction into ethylenediaminetetra-acetic acid tubes and immediately centrifuged; then the plasma was transferred to graduated centrifuge tubes and frozen. The plasma was thawed; the volume was measured and transferred to small Erlenmeyer flasks; and two volumes of ethanol-methylal ( 1: 5 by volume) was added. (5,6-3H) -PGF,a: (0.5 x lo6 c.p.m., specific activity 1.37 mc. per milligram) was added to the mixture to calculate recoveries throughout subsequent purification of the plasma PGF,(u. After stirring for 30 min. at room temperature, the fine precipitate was suction filtered through Whatman No. 42 paper, and the filtrate was evaporated to dryness in vacua. The residue was extracted with chloroform-methanol (2 : 1 by volume) and filtered, and the filtrate was evaporated again to dryness. The flask contents were partitioned between equal volumes (20 ml.) of petroleum ether and ethanolwater (2 : 1 by volume). The petroleum ether was discarded, and the aqueous ethanol was evaporated to dryness, taken up in a small volume of 6 per cent methanol in chloroform, and applied to a Pasteur pipette filled with silicic acid HA to a height of 5.5 cm. held by a cotton wool plug and equilibrated with the same solvent. Elution from the silicic acid was carried out with 5 ml. of 6 per cent methanol in chloroform, 2 x 5 ml. of 20 per cent methanol in chloroform, and

Febl uary 1, 1972 Am. J. Obstet. Gynecol.

finally 5 ml. of 100 per cent methanol. All the radioactivity was eluted with the 20 per cent methanol in chloroform, and this fraction was further purified by preparative thinIayer chromatography on silica gel G plates developed in chloroform-methanol-acetic acid-water (90: 8 : 1: 0.65 by volume). The prostaglandin F band was removed, transferred to a small column fitted with a sintered disc, and eluted with 40 ml. of 90 per cent methanol. The solvent was removed in vacua, and the residue was extracted with freshly distilled ether and 0.025N HCl. The ethereal phase was washed to neutrality, evaporated to dryness, and converted to methyl esters with a solution of freshly prepared and distilled ethereal diazomethane in methanol (9: 1 by volume). The methyl esters were transferred with chloroform-methanol (2 : 1 by volume) to microampuls (total volume 50 ~1) fitted with rubber septa and evaporated to dryness by a stream of nitrogen, and 1 pg of PGF,(w methyl( d3) ester was added. The deuterated PGF,a carrier was found by mass spectrometry to contain 3 deuterium atoms. The ratio of deuterium to protium was better than 1,000 to 1. By first exchanging the freely exchangeable protons of PGF,(u, 3 deuterium atoms could be introduced by deuterated diazomethane. The plasma prostaglandins and carrier were converted to trimethylsilyl ethers by reaction with 10 ~1 of Trisil-Z (Pierce Chemical Co.) and heated for 5 min. at 60’. Half the sample was introduced into an LKB-9000 GC-MS equipped with a column of 3 per cent SE-30 on Chromosorb W maintained at 245’. The range from m/e 415 to 433 was repeatedly scanned every 3 seconds at 20 ev. during the appearance of the peaks of d,-PGF,a! (containing plasma PGF,a) and 3H-PGF,(r which were well resolved on the gas chromatogram. The ratio of deuterated PGFza to the plasma protium form can be obtained from measurement of the peak heights at m/e 426 and 423, respectively, due to the M-(TMSiOH + C,HI1) fragment. With this ratio the proportion of protium to deuterium form can be calculated from a standard line constructed from known quan-

Volllme

112

Therapeutic

N1mlher 3

Table II. Induction

of abortion

by intravenous

infusion

abortion

with

PGF,tr

333

of PGF,(r -.-

Case

No.

Gestation (weeks/days)

Total

dosage (mg.) 72.00 141.00 90.30 93.75 33.25 146.40 96.00 148.60 81.20

Gravity/parity

1 2 3 4 5 6 7 :

10/5 11/2 12/5 13/l 13/4 14/o 14/l 14/6 14/4

3/2 l/O l/O l/O 3/2 l/O l/O l/O

10

15/l

l/O

Mean Mean

Induction-abortion interval (hourq’min.)

80.25

total dosage of PGFza: 93.53 mg. induction-complete abortion interval:

24 hours,

Results

24/45 (49/25) 27/33 30/15 7/55 31/39 28/30 26/04 31/38

Complete Incomplete Complete Complete Complete Complete Complete Complete Complete Complete

25/45 41 min. 140

tities of the protium and deuterium forms of PGF,(u (Fig. 1). The limit of detection by this method is about 3 ng. Thus, when 20 ml. samples were taken, we could detect a concentration down to 0.15 ng. of PGF,a per milliliter of blood. Mean recovery of 69 per cent was obtained for the whole procedure.

r

/*

Results

Abortifacient

efficacy.

The

results

ob-

tained with the 10 patients are summarized in Table II. Nine patients aborted completely. One patient aborted the fetus but failed to expel the placenta after 48 hours and required surgical evacuation. The inductionabortion interval ranged from 7 hours, 55 min. to 31 hours, 39 min. with a mean of 24 hours and 41 min. The interval from incomplete expulsion to complete expulsion ranged from zero to 12 hours, 30 min. with a mean of 3 hours and 24 min. With the unsuccessful case, 22 hours and 45 min. lapsed between initial expulsion and surgical evacuation. Blood loss ranged from 50 to 450 ml. with a mean of 159 ml., and one patient received a transfusion of 2 U. of blood. No other oxytocic agents were used. The total amount of PGF,cY used to induce abortion in the 9 successful cases ranged from 33.25 to 148.60 mg. with a mean of 93.53 mg. In the unsuccessful case, the patient was infused with 141 mg. One patient aborted during the initial infusion period. Another aborted during the 12 hour period following

20

40

ng PGF,,-CH,/pg

60

So

PGFza-CD,

Fig. 1. Standard line relating heights of the mass spectrometric concentration of the protonated methyl ester forms of PGF+

loo

Carrier

the ratio of peak fragments to the and deuterated

initial infusion. The remaining 8 required a reinfusion of PGF,a for a mean period of 5 hours, 40 min. Effect on uterine contractility. Stimulation of uterine contractility was noted in all patients monitored. The initial response was a gradual increase in tone (10 to 4-O mm. Hg) which appeared 7 to 31 min. (mean 18.2) following onset of infusion. The increased tone gradually waned and was replaced by irregular contractions which were of low amplitude and high frequency. Subsequently, these became more orderly, gradually decreased in frequency, and progressively increased in amplitude. Contractions with amplitude as high as 180 mm. Hg were recorded. Basal

uterine

tone

remained

elevated

through-

334

February 1, 1972 Am. J, Obstet. Gynecol.

Gillett et al.

Time Scale l/5 min. 120 mm Hg 60 0

mm Hg

Control Period

35min.

Omin. t Infusion started 25 pg/min.

120 60 0 Infusion lCOyg/min.

mm Hg

120 M) 0 405min. Infusion lOOj~g/min. 6hr. 45min.j

Fig. 2. Record of the effect (Table II): age 30, parity 55 min.

Partial Expulsion 435 min. (7hr. 15 min.)

of intravenous infusion of PGFzo 2, gestation 13 weeks and 4 days,

Table III. Plasma prostaglandins

Sample Preinfusion 50 pg/min. 100 jLg/min. 200 pg/min. 5 min. post *Statistically ~Statistically

(5 hr. 50min.l

vomiting

(ng./ml. (Mean

time intravenous intravenous intravenous infusion different dierent

(p < 0.02) (p < 0.01)

PGFpr plasma) k S.D.)

0.40 0.80 1.43 3.00 0.5,

+ k + + 1.8

0.27 0.23’ 0.56t 1.08t

from from

preinfusion preinfusion

(5) (8) (10) (8)

(2) levels. levels.

out infusion at a level of 10 to 40 mm. Hg. Fig. 2 is a sample record of the uterine contractility in one patient. The mean frequency of contractions, amplitude, and uterine activity (Montevideo units) of all patients monitored calculated for 10 minute intervals every 30 minutes during initial infusion are illustarted in Fig. 3. Plasma PGF,(u measurements. The concentration of PGF,a in plasma before infusion and at various rates of intravenous infusion are summarized in Table III. Preinfusion levels of PGF,a were found to be 0.40 _+ 0.27 ng. per milliliter of plasma. At an infusion rate of 50 pg per minute of PGFza, higher mean values than preinfusion levels were obtained which were statistically significant at p < 0.02. All other infusion rates of PGFza gave significant increases in plasma PGFza (p < 0.01). The data for samples at various time intervals during in-

on uterine activity. induction-abortion

Patient interval

No. 5 7 hr.,

fusion for individual cases are shown in Fig. 4. At the highest dose (200 pg per minute), more pronounced increases in plasma levels were found which correlated well with the observed rise in temperature (Fig. 5). Measurement of plasma levels 5 min. after termination of the infusion showed decreased PGF,a concentrations, but in one case preinfusion values were not found (Table III). These results indicate the rapidity of clearance of prostaglandins from blood by metabolism. In one case in which abortion was completed during the initial PGF,a infusion at the 100 pg per minute rate, a very high plasma level of 8.7 ng. per milliliter was found when the infusion rate was increased to 200 rg per minute (not shown in Fig. 4). Side effects. Side effects were noted in all patients, but no serious complications occurred (Table IV). Nausea and vomiting were only partially alleviated by antiemetics. Local tissue reaction consisted of pain, erythema, and tenderness at the infusion site and was usually trivial but quite marked in 3 cases. Fifty per cent of the patients developed diarrhea, and 20 per cent complained of vasovagal symptoms (feeling of undue warmth or chills). All of these side effects regressed rapidly when the infusion was terminated. Elevation of temperature above 99O and up to 103’ F. was noted in 70 per cent of

Volume Number

112 3

Therapeutic

abortion

with

PGFZ(w 335

Montevideo Units

200

Infusion Rata 1m

Infusion Time (Hours)

Fig.

3. Effect infusion rates. weeks, 1 day.

Table IV.

Incidence

of intravenous Mean values

of side

infusion of PGFza for 8 cases with

on uterine gestational

contractility ages from

and activity at various 11 weeks, 2 days to 15

effects Infusion $1

Side effect Nausea and vomiting Local tissue reaction Pyrexin > 99” F. Diarrhea Vasovagal symptoms

cases. This appeared related to the drug as the incidence was much greater at the 200 pg per minute infusion rate and subsided when the infusion ended. It was not related to dehydration, local tissue reaction, time of abortion, or infection. Fig. 5 illustrates quite dramatically this pyrexial response. A rise in pulse rate accompanied the pyrexia. No changes were noted in blood pressure or respiratory rate. Laboratory tests. No abnormalities in biochemical parameters were encountered in

70 70 30 40 20

sate

(pg/min.) 100 (%I 80 90 30 30 10

Total 200 (%) 90 90 70 40 10

incidence (%I 100 100 70 50 20

blood or urine. The total white cell count was transiently increased in 9 cases (one case not determined) during the infusion period to a mean level of 63 per cent above the preinfusion values (range 14 to 108 per cent). No explanation could be found for this transient leukocytosis apart from the prostaglandin infusion. Other hematologic values were within normal limits. Serum progesterone levels showed no significant decrease during the infusion of PGF,cY. A significant decrease was only ob-

Gillett

February 1, 1972 Am. J. Ohstet. Gynecol.

et al.

asymptomatic bilateral firmed by laparoscopy,

hydrosalpinx,

con-

Comment

Fig. 4. Plasma venous infusion

PGFla levels of PGF2a.

served

the expulsion

after

in 5 cases

during

intra-

of the placenta. Electrocardiogram records. Electrocardiographic changes were seen in 7 cases. In 5, these included slight alterations in the axis, P wave, QRS complex, S-T segment, and T wave. One case showed more marked changes which consisted of prolongation of the Q-T interval, sagging of the S-T segment in Leads 2, 3, and aV,, and negative T waves in V1 through V, which became more pronounced with increasing infusion levels. Another Patient developed occasional premature ventricular contractions at approximately 30 second intervals at the 200 pg per minute infusion level. In none of the patients in the study was there any other evidence of cardiac disturbance or, particularly, myocardial ischemia. Postabortion follow-up. Each patient was seen from 4 to 6 weeks post abortion. Four patients recovered uneventfully. Two cases in which there were elevated temperatures of 100° F. in the immediate postabortion period were diagnosed as endometritis; the patients responded to antibiotic therapy. Four patients, including one who had endometritis, continued to bleed slightly. One of these patients underwent curettage at another hospital, and the other three responded to hormone therapy.* One patient developed *Ortho-Novum, New Jersey.

1 mg.,

Ortho

Pharm.

Corp.,

Raritan,

The success rate for complete abortion found in this study (9 out of 10 cases) compares well with that reported by KarimlO and Karim and Filshie? who used a continuous intravenous infusion of PGF,(u at a dosage level of 50 pg per minute and Wiqvist and Bygdeman13 who varied the intravenous dose up to 360 pg per minute. Direct comparisons of the mean induction-abortion intervals for all the cases reported so far is not possible because of the differences in infusion schedules. Considering, however, that in the present study the infusion was discontinued for 12 hours after the original 12 hour infusion period, whereas in Karim’s study infusion was continuous, the mean inductionabortion interval is not too different (24 hours, 41 min. against 19 hours). The mean total PGF,(u dose to complete abortion was 93.5 mg. which is higher than the 57 mg. calculated from Karim’s results but may be explained by the stepwise dosage increase in this protocol which was designed to study tolerance as well as efficacy. Therefore, on the basis of these comparisons, there appears to be no advantage in infusion schedules of greater than 50 pg per minute. In addition, uterine activity seen during the 50 pg per minute infusion period is comparable to levels seen with abortion induced by intraamniotic saline.14 Nausea, vomiting, local tissue reaction, and diarrhea, even at the 50 pg per minute infusion rate, occurred in a high percentage of cases. In addition, the possibly more ominous changes in the electrocardiogram and in temperature suggestive of myocardial and central nervous effects occurred at the higher infusion and plasma levels. Adaptation of a new mass spectrometricisotope-dilution analytical method applied recently by Samuelsson and associates12 for the determination of prostaglandin E, in blood has enabled for the first time the monitoring of plasma PGF,(r concentrations during infusion in 6 of the 10 cases studied. The tech-

Volume Number

112 3

Therapeutic

104 -

k !+

102-

= I” 2 0

loo-

Initial

Post Initial Infusion

Infusion

abortion

with

PGF?N

337

Post Re-infusion

Re.infusion

--

Temperature PGF,, vgfmin.

9a- ,--40 -25

.‘50

4

I

a

I

12

I

4

I

I

a

I

12

I

I

4

HOURS

I

I

a

I

I

12

L

I

4

I

I

0

1

I

12

4 Complete Abortion

Fig. 5. Record 21,

gravida

of the effect of 1, para 0, gestation

PGF?(r infusion on temperature 14 weeks, induction-abortion

nique is highly specific but requires rather tedious purification methods. We have found that there are low but measurable PGF,cr levels in venous blood in pregnant women. Hamberg and Samuelsson have reported that the concentration of prostaglandin E, is 0.7 ng. per milliliter of blood. Each doubling of the rate of infusion of PGF2(r (Table III) is accompanied fairly closely by a doubling of the plasma concentrations (Fig. 4). Twenty milliliters of blood was needed to determine the PGF,(u levels before infusion and during the lower infusion rates, and this limits the number of samples which can be taken. A specific assay which can be applied to 1 to 5 ml. of blood would be highly desirable. Radioimmunoassay of prostaglandin levels offers some hope, but as yet there are a number of problems in its application to blood determinations. It can be concluded, however, from our studies that plasma PGFp~ levels around 1 ng. per milliliter can initiate and maintain uterine activity. In conclusion, it seems clear that intravenous PGF*LY is an effective abortifacient in the first and early second trimesters through its oxytocic action. Compared with other widely accepted methods for the first trimester (suction, dilatation and curettage),

REFERENCES

1.

2.

Bygdeman, M., Kwon, S. U., Mukherjee, T., and Wiqvist, N.: AM. J. OBSTET. GYNECOL. 102: 317, 1968. Bygdeman, M., Kwon, S. U., Mukherjee, T., Roth-Brandel, U., and Wiqvist, N.: AM. J. OBSTET. GYNECOL. 106: 567, 1970.

(Case No. 6, Table II). interval 31 hr., 39 min.

Age

however, the prostaglandin terminations by the intravenous route are inefficient in terms of time required, expense, supervising personnel, and even success rate. In the second trimester, PGFza administered by intravenous infusion compares more favorably with the commonly used methods (intra-amniotic saand line, hysterotomy) , but the incidence nature of the side effects, seen at an effective dosage level, are sufficient to make us believe that there is little future for PGF,(u as an abortifacient when given by this route. A more direct delivery method is needed if these side effects are to be avoided. Intraand intra-amniotic uterine-extra-amniotic injections will perhaps meet these requirements, and recent reports on these modes of delivery are encouraging.15, lb: Alternately, the use of other prostaglandins or prostaglandin analogues which possess greater discrimination between their oxytocic effect and side effects may obviate this problem. The authors wish to thank Miss S. Myles, R.N., Dr. J. Patrick, and Mrs. K. Rostworowska for

skilled

technical

assistance,

and

Dr.

0.

Mamer of the Mass Spectrometry Unit, Royal Victoria Hospital, Montreal, for assistance in the mass spectrometric studies.

3. 4.

5.

Wiqvist, N., Roth-Brandel, U., and Bygdeman, M.: Int. J. Obstet. Gynecol. 8: 165, 1970. Karim, S. M. M., Trussel, R. R., Hillier, K., and Patel, R. C.: J. Obstet. Gynaecnl. Br. Commonw. 76: 769, 1969. Karim, S. M. M., Hillier, K., Trussel, R. R., Patel, R. C., and Tamusange, S.: J. Obstet.

338

6. 7.

Gynaecol. Br. Commonw. 77: 200, 1970. Roth-Brandel, U., and Adams, A.: Acta Obstet. Gvnecol. &and. 49: 9. 1970. ISUDDI. 5.) Karim,‘S. M. M., and Filshie, G. M.:‘Lancet

1: 157, 1970. 8. 9. 10. 11.

February 1, 1972 Am. J. Obstet. Gynecol.

Gillett et al.

Embrey, M. B.: Br. Med. J. 2: 258, 1970. Bygdeman, M., and Wiqvist, N.: Ann. N. Y. Acad. Sci. 180: 473, 1971. Karim, S.: Ann. N. Y. Acad. Sci. 180: 483, 1971. Embrey, M.: Ann. N. Y. Acad. Sci. 180: 518, 1971.

12. 13. 14.

15. 16.

Samuelsson, B., Hamberg, M., and Sweeley, C. C.: Anal. Biochem. 38: 301. 1970. Wiqvist, N., and Bygdeman, M.: Lancet 1: 889, 1970. Turnbull, A. C., and Anderson, A. B. M.: J. Obstet. Gynaecol. Br. Commonw. 72: 755, 1965. Embrey, M. P., and Hillier, K.: Br. Med. J. 1: 588, 1971. Karim, S. M. M., and Sharma, S. D.: Lancet 2: 47, 1971.