Electrometric timing of human ovulation

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acid obtained after acid hydrolysis is referred to and offered as a means of eliminating the source of error experienced with the method Venning.“” RlWERESCES

Rubenstein, B. B.: Enllocrinolog27: SX:, 1Ylll. Seymour, F. I.: J. A. M. A. 112: 1X17, 19Xl. Mirscbrr. K.. Fischer. W. H.. and Tschoon E.: Nature 142: 1%. 1938. ~~euverss~vvn~ J. I’.. ‘C’ollins.’ V. S.. ant1 ~~Villiams. W. L.: I’roc. Sot. EXDCT. I Biol. & dM&l. 41: &Z, l!I:&. Leathem, J. IX., ant1 Crafts, R. C.: hat. Rev. 76: 90, 1940. Wells, J. A., and Greene, 1~. R. : Am. J. Physiol. 126: 652, 19:X). Klein, M., xnd Yarkes, A. S.: IVoc. Roy. SOP. Lontlon n. 121: 574, 1937. Rohson, J. M.: Quart. J. Exlw. Physiol. 26: Xi.5, 1927. Zuck, T., and Duncan, D. R. I,.: i\.\r. J. OBST. & GYNW. 38: 310, 1939. Burr? H. R., Musslman, L. K., Barton, .I). B., am1 Kelly, N.: Science 86: 319, 19:47. _.

Rubenstein, B. B.: Am. J. Physiol. 129: 4.51, 1940. Samurls. J.: Surz.. Gvnet:. & Old. 67: fiOR. 19X% Venning, E. II., ii~i B’roww, .J. 8. II.: Endbcrinology . 21: ill, 19::7. PnrwCcolnou, G. r\‘.: Am. J. hat. 52: 519. 1933. S&r. E. : Proc. Sot. Exrrcr. Bid. & Med. 43: 51)l. 19Ul Shorr; E.: Science 91: 5+?1, 1940. Rubenstein, B. B., ant1 Dunran. 1). R,. L.: Enclocrinolozv 28: 911. 19-&l Hhorr, E: I’ror. Hoc. Exper. Biol. & MelI. 43: 501, 19-40. Venning, E. H.: J. Biol. C’l~em. 126: X5, 19:!8. Rubmstein, B. B.: Person:11 communic:ttion. Hamlllen, E. C., Ashley, C’., anal Ilaptist. RI.: En(locrinoIo~q24: 1, 1939. Westphal, V., and Burton, C. L.: 1’1.0~. Sot. Esper. Bid. & Med. 42: 749, Marker, R. E., and Hartman, (‘. G.: J. Hiol. (‘hem. 133: 5t’R, 1940. Vcnning, E. H., ant1 Brownc, .J. S. L.: Entlocrinology 27: 707, 19411. Hechter, 0.: Prw. Sot. Esper. Biol. & Med. 49: 399, 1942. Allen, \Y. JI.. ant1 Viergiver, I?.: .J. Binl. (‘hem. 141: 8:X7, INl.

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1939.

NVESTI(L4TORS have long sought an adequate method for determining with certainty the time of ovulation in animals and in man. The usual methods of mating at selected times and the examination of the generative tract at laparotomy have given us a good deal of valuable information. In man these procedures have been time-consuming and not altogether satisfactory, hence the report, by Burr, Hill and Allen4 that ovulation in the rabbit was accompanied by significant modifications of an electrical patt.crn in the intact animal, offered a new procedure fraught with many possihilit.ies. The findings were confirmed h,v Rehoul, Friedgood and Davis7 and extended by A1tman.l Subsequently Burr, Musselman, Barton and Kellcy” reported an electrometric change in the *Received

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publication,

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human being with confirmation at laparotom?-. In this instance a recently ruptured follicle was observt~l and a l)c)rtion of Ihe ovary conlaining it was removed and studied mic~1~os~oI,ic~all~.Rock, Rc~boul and Wigger9 extended these findings to larger grouI) and confirnictl the csI)erimcnt, although there were somt’ tiiscrcpancics. il

The apparatus used in the procedure was a Burr-Lane-Nims 1X microvoltmeter with a Cieneral Electric photoelectric recorder as a recording galvanometer (Fig. 1) . The paper speed was one inch in five minut,es and its sensitivity one millivolt per division. The zero baseline was in the center of the chart. Deflections of the pen to the right indicated positive polarity of the input lead to the grid of the microvoltmeter; deflections to the left, negative polarity of this lead. Contact of the microvoltmeter with the patient was matlc by a pair of nonpolarizable reversible silver-silver chloride clcctrodes immersed in physiologic salt solution. From t.hc chambers containing these electrodes, rubber t,ubes were led to the patient. The one from t,he ground electrode terminated in a sponge rubber disc which could be strapped to the ankle. This

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constituted the reference or cold electrode and it.s exact location on the surface of the body did not seem to be important. The ankle was chosen for convenience. The other electrode chamber was connected to the paGent by means of a rubber tube, the outer end of which was cemented The cavity of the into the dome of a thin rubber cervical diaphragm. diaphragm was packed with cotton sat,urated with normal salt solution Both tubes were filled with and was readily placed over the cervix. saline, taking care to see that there were no bubbles of air in the lumen of the tubes or between the tubes and the surfaces of the body. In practice, the patient lay comfortably on an examining table for periods varying in time from fifteen minutes to more than an hour. No discomfort or pain was encountered at any time. It should be noted that this technique made it possible to record electrical changes in the intact organism. Hence, at all times, there was a minimal disturbance in normal physiologic processes. The use of silver-silver chloride electrodes gave assurance of a faithful record of t,rue changes of potential difference without the artifacts of contact potential, such as are encountered when metal is brought directly into contact with hod- surfaces. In practice, these elect,rodes are stable and introduce no artificial e.m.f.‘s in to the external circuit. While the mic~~ovoltmcter responds adequately t.o voltage changes at wide differences in frequencies, the recording galvanometer used imposes certain limitations on the records. The period of the galvanometer used is approximatclv three seconds. As a result, it did not respond readily to relatively rapid changes, such as accompany the heart, nerves, or striated muscle. Fortunately, howcvCr, nonstriated muscle contractions produce electrical changes of much lower frecluency. Faithful records, therefore, could be obtained. The cases were selected from the patients of a. gynecologie practice and the studies were carried out in the ofice. The procedure was employed as a means of improving on the techniques previously used (Langman and Burr”) in an electrical study of uterine mobility during the menstrual cycle. While interesting data on this point were obtained, it was overshadowed by an unexpected finding, which it, is the purpose of this report to present. AS.iI,YSES

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DIS(‘l’SSIONS

Approximately 150 records have been taken from 7 women on the majority of the days of the menstrual cyvcle. The studies showed that most commonly throughout the menstrual cycle the cervix was positive to the ankle from 5 to 25 mv. In 9 of the 14 cycles studied, the cervix became negative, an equivalent amount on at least one day of each rycle. In four of these, the cervix rcmaincd negative for two days. The days of the cycle on which the negativity appeared were the third, sixth, seventh, eighth, thirteenth, fourteenth, eighteenth, or twenty-first. In five cycles eit,her no negat.ive shift occurred or it was eqnivoeal. The general distribution of these results is shown in Fig. 2. It will he noted that there are many gaps in each cycle, due to the necessary exigencies of an office practice. In a number of instances the recorded potential difference was so near the baseline that it was called equivocal and is marked on the chart with a question mark.

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ing. Two days later the records showed the same kind of phenomena but by t.he thirty-second day they disappeared. At this time the breasts became tender and the patient, gave subjedive evidence of pregnancy. This was confirmed by the Aschheim-Zondek t,est and by subsequent history (Fig. 3, A, B, C). The situation in this particular patient was important because, although the patient had been married for several years, mating had been infertile. On examination this was shown to be due to azoospermia. Artificial insemination with donor sperm was resorted to on this one occasion only, in order to produce the desired pregnancy. The circumstances, therefore, acted as a very adequate control for the experiment. The fact that artificial insemination was successful suggests that the negative shift occurred at the time of ovulation, as it does in the albino rat.

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B Fig. 3.-Tracings of photoelectric cervix positive. B, Fourteenth Fifteenth day after insemination day of A. B. showing electrical

recordings. A,, The ninth day of D. H. showing day of same patlent showing cervix negative. C. showing electrical activity of the cervix. D, Ninth activity of the cervix following hormone injection.

It is noteworthy that the regular and rhythmic changes in electrical potentials described above appeared on the day when the normal menses would have occurred. The character of these changes is interesting, because they are very similar to the kind of phenomena which have been recorded in the generative tract of the albino rat during estrus. Boling has shown that these rhythmic phenomena can he induced in spayed animals by the administration of hormones. Moreover, it is generally believed that some two weeks after fertilization there is a sharp rise in the hormone level in the circulating body fluids. The evidence here presented indicates the probability that the impact of chemical agents on the generative tract produced significant changes in the electrical pattern. Since the general muscular activity of the ut.erus, as reflected in changing potentials, is an important element in the physiology of the generative tract, it should be noted that all the electrical records showed a higher level of activity as early as the sixth and as late as the twenty-first day of the cycle. This fits in very well with previous findings. An additional interesting observation was that on the fourth or fifth days prior to the onset of a succeeding menses,the positivity of the cervix increased

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to as much as 30 or 35 mv. and usually remained there until the actual onset of bleeding. With the onset 0; the flow, the voltage difference dropped to more nearly the normal 15 mv. The history of Patient, ‘i (A. B.) is also of’ considerable interest. Although apparently a healthy youn, v woman, wit.h a regular t.wenty-eiglitday cycle, and married for several years, mating had been infertilt,. On examination it was found that the uterus was considerably smaller than usual, approaching the infantile t,yptJ. However, a number oL’ recortls were taken as is shown in I?@. 2, and a negative shift appeared on the sixth and seventh days of the second cycle. Immediately following this, artificial insemination, using husband’s sperm, was carried out. Twenty-one days later. when mt’nses would have normally appeared, nonc occurred. However, rnenst’s did appear thleven days lat.er. On the day before the onset. of mensrs, t hc* ljatient, as an air raid warden, had found it necessary to run up and down stairs frequently. it is not impossible, therefore, that, fertilization might havca occurred but with subsequent early ilbOrt ion. In an attempt to build 111)the ntrrinc mechanism in the subsequent cycle, estradiol benzoatc~ ( 10,000 R.I’. ) was administered on the eighth day. Twenty-four hours later the record shown in Pig. 3, D was ohtained. This characteristic record is almost identical with that obtained from Patient, 6 (I). Ii.). fifteen days after suctvxd’ul insemination. 111 both instancrs the rrc*ortfs are very similar to those obtained during t ht! Twenty-four ~~OUYS period of heat in the albino rat (Boling and others”). later the same phenomrnon was obscrvcti although the magiitude of the On this, rhythmic changes was reduced and the frequent? increased. the tenth day, the patic>nt was givc,n another injection of the same amount and again on the thirteenth day. Following the last injectiotr the rhythmical changes wcrc ljresent bat much reduced in frequency and amplitudr. Two cycles later, cstradiol bcnzoatr (4,000 R.V.) was given on thr third da,- of the cylc. For thcj succ.eeding four or five days, there wcrt: modcrate signs of uterine act.ivit,y alld on Ihe ninth day a second injection of &radio1 was given. Twenl-y-four hours la& the cervix was negative and showed rh\-thmical changes of low magnitude but relatively A&i&~1 insemination wilt1 husband’s sperm \trils high frccluency. carried out and frequent records titketl up to t llt, cight,eenth da?. ,I1 t,he time when subsequent menses were due, t lit patient gave sublective signs of pregnancy with swellin, 0 and tendernt,ss of 1.11~breasts. ,111 Aschheim-Zondek test, lrowcver, made a few day9 later, was negative and cvt~ntually menses appeared nine days late. It 1s possible that here again fertilization occurred with subsequent early abortion. The apparent absence of correlation between negative shift of thrb cervix and ovulation is important for it indicates that thrrc is still a good deal to bc learned about electrical phenomena associattd wit.11 the physiology of thcl Fettt’l’;ltive tract. Many more carefully cont1~ollt~ti studies must hta carrttd ollt before the final answer can be d&covered.

The implications of the findings of’ this study are of considerable interest. There are obviously a good many variables involved in the physiThis is also true of t,he single event of ology of the generative tract. The fact that it was possible to get at least one positive ovulation. correlation makes it worthwhile to extend these studies to as many pa-

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tients as is possible. The number of volunteer patients in an office practice is necessarily limited, so that it is desirable that the technique be employed as widely as possible. Attention, however, must be called to the fact that while the procedure is relatively simple, certain safeguards against artifacts must be employed. It should be noted that the hot lead goes to the cervix and not to the wall of the vagina. That this is important is suggested by some findings on t,hc electrophysiology of the generative tract in the monkey where there is a very sharp gradient between the vagina and the OS,wit,h a polar reversal. Unsuspected grounds on the patient are a possible source of error. Air bubbles in the leads to the electrodes are another. Interference from other elccbrical devices is occasionally met with but can usually be recognized without difficulty in the photoelectrical record. The procedure is simple enough, however, so that the studies are being currently continued. A second aspect of the findings is the pronounced effect of the injection of a hormone upon the electrical record. This corroborates in the human experimental data derived from the albino rat. They suggest the possibility of studying t,he effect of a variety of hormones upon gcnerative physiology. The technique has the advantage of being employed on the intact organism, interfering little with the normal physiology. It is not impossible, therefore, t,hat it can be utilized as an effective adjunct to other procedures in the study of endocrine physiology. This report is presented not as a final exposition but in the hope that it will stimulate others to extend these studies. REFEREKCES

1. Altman, Margaret: J. Camp. Psychol. 31: -173, 1941. 2. Boling, J. L., Barton, D. S., and Burr, H. P.: hnat. Rec. 76: Supplement No. 3, 8, 1940. 3. Boling, J. L., and Burr, H. S.: Anat. Rec. 79: Supplement No. 2, 9, 1941. 4. Burr, H. S., Hill, R,. T., anal Blien, Edgar: Proc. Sot. Exper. Biol. & Med. 33: 109, 1935. 5. Burr, H. S., Musselman, L. K., Bart,on, D. S., and Kelly, N. B.: Yale J. Biol. & Med. 10: 155, 1937. 6. Lang-man, Louis, and Burr, H. 8.: AM. J. OBST. & GYNEC. 42: 59, 1941. 7. Reboul, J., Friedgood, H. B., and Davis, H.: Am. J. Physiol. 119: 357, 791X7. 8. Rock, John, RebouI, J., and Wiggers, H. C.: New England J. Med. 217: 654, 1937. 9. Rock, John: New England J. Med. 225: 910, 1941. 108 EAST

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STREET ADDENDUM

We wish to report observations completed on two additional women since submitting the original manuscript for publication. They substantiate our previous findings and in the case of Patient L. B., add one more positive correlation with ovulation time to our series. PATIENT 8.-M. J., aged 34 years, had a history of sterility for two and one-half years; menstrual cycle for the past year had been regular, thirty-one to thirty-two days. On the eighteenth day of the cycle the cervix was recorded as 5 to 15 mv. negative, having previously been posi-