The prediction of ovulation is necessary for oocyte aspiration in a spontaneous cycle and can be reliably achieved only by measuring luteinizing hormone (LH). Since radioimmunoassays of LH take too long for repeated measurements on the same day, we evaluated the possibility of adapting a rapid and fully automated assay of serum LH for use with urine samples. The study group comprised spontaneously ovulating women (38 cycles) who requested artificial insemination. Their serum oestradiol (E2) levels, ultrasound profile (US) and thrice daily urinary LH levels were determined from day 10 of their menstrual cycle. These patients were followed until US signs of follicular rupture were recorded. In all patients, a well-defined LH peak was measured in the urine. This peak lasted 12-15 h and was followed in 35 cycles (no US available for 3) by follicular rupture 9-51 h later. The data were grouped according to the time of the LH peak on day 0. Patients experiencing an LH peak between 0300 h and 0700 h on day 0 had significantly lower levels of E2 on day 0 compared to those with an LH peak between 2200 h and midnight. This is due to the fact that in the patients with an LH peak between 0300 h and 0700 h, E2 levels were already decreasing (from day 1 to day 0), whereas in those with the LH peak between 2200 h and midnight E2 levels were still increasing on the morning of day 0. We conclude that the 30-min IMX LH assay is a reliable, rapid and readily acceptable method for measuring urinary LH and for the prediction of ovulatio

Bianchi, P.G.

Bischof, P.

Campana, A.

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Human Reproduction vol.6 no.4 pp.515-518, 1991Comparison of a rapid, quantitative and automatedassay for urinary luteinizing hormone (LH), with an LHdetection test, for the prediction of ovulationP.Bischof, P.G.Bianchi and A.CampanaClinic of Infertility and Gynaecological Endocrinology, Departmentof Obstetrics and Gynaecology, University of Geneva, 1211Geneva, Switzerland'To whom correspondence should be addressedThe prediction of ovulation is necessary for oocyte aspirationin a spontaneous cycle and can be reliably achieved only bymeasuring luteinizing hormone (LH). Since radioimmuno-assays of LH take too long for repeated measurements on thesame day, we evaluated the possibility of adapting a rapidand fully automated assay of serum LH for use with urinesamples. The study group comprised spontaneously ovulatingwomen (38 cycles) who requested artificial insemination. Theirserum oestradiol (E2) levels, ultrasound profile (US) andthrice daily urinary LH levels were determined from day 10of their menstrual cycle. These patients were followed untilUS signs of follicular rupture were recorded. In all patients,a well-defined LH peak was measured in the urine. This peaklasted 12-15 h and was followed in 35 cycles (no US availablefor 3) by follicular rupture 9-51 h later. The data weregrouped according to the time of the LH peak on day 0.Patients experiencing an LH peak between 0300 h and 0700 hon day 0 had significantly lower levels of E2 on day 0compared to those with an LH peak between 2200 h and mid-night. This is due to the fact that in the patients with an LHpeak between 0300 h and 0700 h, E2 levels were alreadydecreasing (from day 1 to day 0), whereas in those with theLH peak between 2200 h and midnight E2 levels were stillincreasing on the morning of day 0. We conclude that the30-min IMX LH assay is a reliable, rapid and readilyacceptable method for measuring urinary LH and for theprediction of ovulation.Key words: luteinizing hormone/IMX-assay/ovulation predic-tion/ultrasonographyIntroductionThe prediction and detection of ovulation are often critical in thetreatment of infertility. Many different parameters have been usedto predict ovulation including basal body temperature (Moghissi,1976; Lenton et al., 1977), cervical mucus and circulating levelsof luteinizing hormone (LH), oestradiol (E2) and progesterone(P) (Kerin et al., 1981; Collins, 1985; Bryce et al. 1982).Measurements of vaginal impedance (Albrecht et al., 1985) orrecording of Mittelschmerz flower abdominal pain at mid-cycle)(O'Herlihy et al., 1980) have been described as preovulatorysigns with a rather poor degree of ovulation prediction.Follicular size and recent ovulation can be detected byultrasonography with great accuracy (Renaud et al., 1980; Bryceet al., 1982). This method is not infallible in detecting ovulationsince it shows follicular rupture and not egg release (Elkind-Hirsch et al., 1986). Determining follicular size by ultrasoundin order to predict ovulation is highly inaccurate since the perioduntil ovulation varies greatly among women with equal follicularsize (Renaud et al., 1980; Kerin et al., 1981; Bryce et al., 1982).Quantitative (Kerin et al., 1980) or semiquantitative (Lloydand Coulam, 1989) measurements of urinary LH have been foundsuperior to most other methods used to predict ovulation. Sinceconventional methods of radioimmunoassay of LH are too longfor repeated measurements during the same day, we evaluate herethe possibility of adapting a rapid (30 min), quantitative and fullyautomated assay of serum LH for use in urine samples.Materials and methodsPatientsA total of 32 spontaneously ovulating women between 28 and37 years of age were included in this study. All patients attendedour infertility clinic for artificial insemination. None receivedany medication or hormonal therapy during the evaluated cycles(n = 38). Blood samples were taken daily between 0800 h and0900 h onto ethylenediaminetetraacetic acid (EDTA, 2 mg/ml)starting on day 10 of the cycle until ovulation (see below). Duringthis period, the patients were asked to collect three urine samplesper day (morning, afternoon and evening) and to record the timeof sampling.Ultrasonographic monitoringUltrasonographic imaging of the ovaries was performed with aToshiba Sonolayer-L fitted with a 5 MHz vaginal transducer(Medical System, Aubonne, Switzerland). Transvaginal ultra-sonographic scans were begun on day 10 of the menstrual cycleand continued daily until follicular rupture was documented.Ovulation was defined as a reduction in follicular size of at least5 mm (Check et al., 1990). Ultrasonographic monitoring wasalways performed between 0800 h and 0900 h. Follicular sizewas recorded by measuring two right-angle diameters for eachfollicle and then taking the mean of both diameters.Hormone assaysE2 was measured daily in the plasma samples using a commer-cially available radioimmunoassay (Biomerieux, Marcy L'Etoile,France). LH was measured daily in urine samples by an© Oxford University Press 515P.Bischof, P.G.Bianchi and A.Campanaautomated microparticle enzyme immunoassay for serum LH(IMX, Abott, Cham, Switzerland) using a mouse anti-human LHmonoclonal antibody. The sensitivity of this assay for serum LHwas determined by the manufacturer at 0.5 mlU/ml and the co-efficient of variation at 3.6%. The day of the urinary LH peak(highest value) was designated day 0.In order to compare the IMX assay with a standard ' 'Ovulationtest", 103 consecutive urine samples were obtained from 43patients. LH was measured with the IMX and with a semi-quantitative LH detection assay (sensitivity 50 IU/1, LH colour,Organon, Oss, The Netherlands). All the LH detection assayswere judged by the same technician. Results were evaluatedstatistically by Student's f-test for paired or unpaired variables,when appropriate, using the Fastat program (Systat, Evanston,IL, USA) on a Macintosh IICX computer.ResultsValidation of the urinary LH assayIn order to evaluate the accuracy of our assay and a possiblematrix effect, a serum-based sample routinely used as an internalcontrol and containing a known concentration of LH was seriallydiluted with urine from a male volunteer and measured on theIMX (Table I). When the serum-based sample was diluted 1:2Table I. Effect of urine on the measurements of LH by the IMX assayDilution LH mlU/mlSerum-based controlMale urineUndiluted1:21:41:81 161:32Undiluted124.90114.7121.8122.7127.0122.91.96-Einary LH60 -50 -40 -30 -i20 "l10 -o -Fffert nf Stnrap^ a t 4 nC^ "1 1 1 1 10 1 2 3 4 5 6DaysFig. 1. Effect of storage at 4°C on the measurements of urinaryLH by IMX assay.516with urine, the measured level of LH was lower than that foundin the undiluted control. Higher dilutions of the control samplegave LH values which were indistinguishable from the undilutedserum control (Table I).The effect of storage on the IMX LH determination was alsoinvestigated. As shown in Figure 1, urine samples could be storedup to five days at 4°C without significant loss of immunoreactiveLH. In contrast, freezing and thawing dramatically reduced themeasurable concentration of LH (results not shown).Table II compares the results obtained with the quantitativeIMX urinary LH assay with those of the LH colour assay. Thecorrelation between the two assays was high (n = 103,r = 0.688, P < 0.001) but an important overlap was observedTable II. Comparison8 betweenLH colourNegativeBorderline11PositiveIMX LH,n621922IMX andIU/1Mean8820.851.1LH colour assaysSD6.06.120.4(n = 103)Range(0.5-28.6)(10.3-36.8)(17.2-95.2)V = 0.688, P < 0.001.bBorderline: colour between a positive and a negative test.TIME OF URINARY LH PEAK ON DAY 03 to 7 AM ^ iu , H J , „ vu ,2 PMFig. 2. Urinary LH levels, serum E2 and follicular size measuredonce a day at 0800 h to 0900 h from day 2 to ovulation.represented according to the time of the LH peak on day 0.Semiquantitative assay for detection of urinary LHTable III. Urine and blood parameters of the 38 spontaneous cycles according to the time of the LH peakUrinary LH peak between:Urinary LH at the peak (IU/1)Serum E2 on day 1 (pg/ml)Serum E2 on day 0 (pg/ml)Difference in E2 between day 1 and day 0 (pg/ml)Follicle size on day 0Unpaired Student's Mest. a vs b, NS; a vs c, P < 0 03; d vs e, P < 0.05; d vs f, P < 0.001.Paired Student's /-test; d, P < 0.01; e, NS; f. P < 0.001.'Ei and follicle sizes as measured once a day between 0800 h and 0900 h.0300n12121212IIh to 0700 hMean54.3251.8207.8a-23.2d21.2SD25.788.683.125.3± 1.41400 hn1312131213to 1900 hMean48.6210.9225.8b20=19.7SD14.078.295.932.62.22200 hn13121312to midnightMean59.9259.3304.6c16.1f20.3SD30.798.2119.69.43.1between negative and positive LH colour tests. The highest valueobserved for a negative test was 28.6 IU/1 and the lowest valuefor a positive test was 17.2 IU/1. About 20% of the colour testsdeveloped a colour which was between a positive and a negativeresult and the LH values ranged from 10.3 to 36.8 IU/1 in theseborderline samples (Table II).Urinary LH and foUicular ruptureAll the 38 cycles studied had a well-defined LH peak in the urine(highest value at least twice the basal level for each patient intwo successive samples). These cycles were divided into threegroups according to the time at which the highest urinary LHvalue had beenVecorded on day 0: 0300 h-0700 h, n = 12;1400 h-1900 h, n = 13; 2200 h-midnight, n = 13 (Figure2). The mean concentration of LH peaks did not differ betweenthe groups (Table m), the levels ranging from 21.0 to 133.0 IU/1.Post-LH peak ultrasound scans could not be performed in 3patients but in all the remaining 35, foUicular rupture wasdocumented at ultrasonography. In the group of patients with aurinary LH peak between 0300 h and 0700 h on day 0, foUicularrupture was seen in one patient on the same day (Figure 2),i.e. about 4 h after the highest LH concentration in the urine(63 IU/1) had been detected. This patient had, however, anelevated LH concentration(52 IU/1) in the urine sample collectedin the evening (2230 h) of day - 1 . In all the other patients,foUicular rupture was seen on day 1 or 2 after the LH peak. Thetime between the urinary LH peak and foUicular rupture (asdocumented by daily ultrasound scans) varied between 9-51 hwith no significant differences between the groups (Figure 3).Serum E2 and foUicular ruptureMean serum E2 concentrations were similar for all three groupson day 1. Day 0 E2 levels in the group of patients with a urinaryLH peak between 0300 h and 0700 h were significantly lowerthan those in the group with a peak between 2200 h and midnight(P < 0.03, Table m). The difference in E2 between day-1 andday 0 was statistically significant between the groups (P < 0.05to P < 0.001, Table HI): the patients with the LH peak between0300 h and 0700 h showed a significant reduction in E2 fromday - 1 to day 0 (-23.2 pg/ml, P < 0.01, Table III) whereasthe patients with an LH peak between 1400 h and 1900 h hadunchanged E2 levels and those with the LH peak between2200 h and midnight showed a significant increase in E2concentrations ( + 16.1 pg/ml, P < 0.001, Table m). This wasTIME FROM URINARY LH PEAK TO FOLLICULAR RUPTURE30c 20^ 101•r c c io Ruptured Follicle el US• Unruptured Follicle at US• Missing 2nd USo • n OD"o" •"Patients with LHpeak from 3-7 AMPatients with LHpeak from 2-7 PMPatients with LHpeak from 10-12 PtiFig. 3. Time between urinary LH peak and ultrasonic evidence offoUicular rupture.because the patients with an LH peak between 0300 h and 0700 hexperienced their highest E2 level on day 1 (11 out of 12patients) whereas all patients with an LH peak between 1220 hand midnight had their serum E2 peak in the morning of day 0.It is interesting to note that the foUicular size on day 0 was similarin all patients (Table III).DiscussionIn clinical practice, semiquantitative assays of urinary LH arewidely used to predict ovulation. Usually the patients areinstructed to use a dipstick method daily to test their morningurine (O'Herlihy et al, 1980; Lloyd and Coulam, 1989; Grinstedet al, 1989; Paz et al, 1990). This approach to ovulationprediction has been found superior to many other methodsincluding assays of serum LH or E2 (Grinsted et al, 1989). Inour hands, rapid and quantitative urinary LH measurementsproved to be superior to the dipstick method. By sampling urinethrice daily and evaluating LH quantitatively, the precise timingof the preovulatory LH surge and peak is obtained. As shownin Table III, a strict time-dependent correlation between serumE2, evidence of foUicular rupture and urinary LH peak isobserved.According to the present data, an increased level of urinaryLH can be observed over a period of 12 to 15 h so that one might517P.Bischof, P.G.Bianchi and A.Campanaexpect a dipstick method with a sensitivity of 50 IU/1 used onceper 24 h to miss a certain number of LH peaks. The precise timingof LH secretion is of paramount importance for oocyte aspirationin spontaneous cycles. It is, however, debatable as to whethersuch a precise timing of the LH peak is necessary for artificialinsemination. This is particularly true if one takes into accountthe lack of knowledge about the survival rate of spermatozoa inthe female genital tract. The rapidity, the ease and the non-invasiveness of the LH measurement described here would arguein favour of its widespread use.Compared to the LH colour technique, the quantitative methodprobably underestimates the real concentration of LH in the urinesince LH colour-positive samples (sensitivity 50 IU/1) haveregistered only 17 IU/1 of LH with the quantitative assay. Thismight be because the IMX uses a monoclonal antibody, raisedagainst serum LH (since this method was designed for serumLH), which probably only partially recognizes urinary LH. Inthe present study, follicular rupture was documented byultrasound scans. However, since these have only been performedon a daily basis, our estimation of the time elapsed between theLH peak and ovulation is probably over-estimated by severalhours. Previous investigations described the onset of the urinaryLH surge to occur beween 12 and 44 h before follicular rupture(O'Herlihy et al., 1980; Singh et al, 1984; Vermesh et al,1987). This is in keeping with the interval observed in the presentstudy (9—51 h) between the LH peak an follicular rupture.In conclusion, the IMX assay of urinary LH proved to be areliable method of ovulation prediction which is readily acceptedby the patients and avoids the need for frequent venepuncture.Furthermore, the assay offers the advantage of being fullyautomated and very rapid, allowing 24 samples to be processedwithin 30 min.radioimmunoassay for luteinizing hormone to detect onset ofpreovulatory LH surge. Lancet, ii, 430-431.Kerin,J.F., Edmonds,D.K., Warnes.G.M., Cox.L.W., Seamark,R.F.,Mattews.C.D., Young,G.B. and Baird.D.T. (1981) Morphologicaland functional relationships of graafian follicle growth to ovulationin women using ultrasonic laparoscopic and biochemical measure-ments. Br. J. Obstet. Gynaecol, 88, 81-88.Lenton.E.A., Weston.G.A. and Cook.I.D. (1977) Problems in usingbasal body temperature in an infertility clinic. Br. Med. J., 1,803-805.Lloyd,R. and Coulam.C.B. (1989) The accuracy of urinary luteinizinghormone testing in predicting ovulation. Am. J. Obstet. Gynecol.,160, 1370-1375.Moghissi.K.S. (1976) Accuracy of basal temperature for ovulationdetection. Fertil. Sterii, 27, 1415-1417.O'Herlihy,C, Robinson.H.P. and De Crespigny,L.C. (1980) Mittel-schmerz is a preovulatory symptom. Br. Med. J., 280, 986-989.Paz,G., Yogev,L., Gottreich,A., Rotem,R., Yavetz.H. andHomonnai,Z.T. (1990) Determination of urinary luteinizing hormonefor prediction of ovulation. Gynecol. Obstet. Invest., 29, 207-210.Renaud,R.L., Macles,J., Dervain.I., Ehert.M.C, Aron,C,Plas-Roser,S., Spira,A. and Pollack,H. (1980) Echographic study offollicular maturation and ovulation during the normal menstrual cycle.Fertil. Sterii., 33, 272-278.Singh,M., Saxema,B.B. and Rathnam,P. (1984) Clinical validation ofenzyme immunoassay of human luteinizing hormone (hLH) in thedetection of the preovulatory luteinizing hormone (LH) surge in urine.Fertil. Sterii., 41, 210-213.Vermesh,M., Kletzky.O.A., Davajan,V. and Israel,R. (1987)Monitoring techniques to predict and detect ovulation. Fertil. Sterii.,47, 259-264.Received on August 31, 1990; accepted on November 26, 1990AcknowledgementsThe authors wish to thank C.Gruffat for technical help and M.Gharbifor typing the manuscript.ReferencesAlbrecht,-B.H., Fernando,R.S., Regas.J. and Betz,G. (1985) A newmethod for prediction and confirming ovulation. Fertil. Sterii., 44,200-207.Bryce,R.L., Shuter,B., Sinosich,M.J., Stiel.J.N., Picker.R.H. andSaunders,D.M. (1982) The value of ultrasound, gonadotropin andestradiol measurements for precise ovulation prediction. Fertil. Sterii.,37, 42-47 .Check,J.H., Adelson.H.G., Dietterich,C. and Stern,! (1990) Pelvicsonography can predict ovum release in gonadotrophin-treated patientsas determined by pregnancy rate. Hum. Reprod., 5, 234-236.Collins,P.W. (1985) Hormonal indices of ovulation and the fertile period.Adv. Contracept., 1, 279-283.Elkind-Hirsch,K., Goldzieher,J.W., Gibbons,W.E. and Besch,P.K.(1986) Evaluation of the Ovustick urinary luteinizing hormone kit innormal and stimulated menstrual cycles. Obstet. Gynecol., 67.450-456.Grinsted,J., Jacobsen,J.D., Grinsted.L., Shantz,A., Stenfoss.H.H. andNielsen,S.P. (1989) Prediction of ovulation. Fertil. Sterii, 52,388-393.Kerin.J.F., Warnes,G.M., Crocken.J., Broom.T.G., Ralph,M.M.,Mattews.C.D., Seamark.R.F. and Cox.L.W. (1980) 3-Hour urinary518

Comparison of a rapid, quantitative and automated assay for urinary luteinizing hormone (LH), with an LH detection test, for the prediction of ovulation

Comparison of a rapid, quantitative and automated assay for urinary luteinizing hormone (LH), with an LH detection test, for the prediction of ovulation

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