JACQUES TESTART, PH.D.** RENE FRYDMAN, M.D.* MARIE CLAUDE FEINSTEIN, PH.D.t ALAIN THEBAULT, M.D.* MARC ROGER, M.D.t ROBERT SCHOLLER, M.D.

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1 FERTIUTY AND STERILITY Copyright " 1981 The American Fertility Society Vol. 36, No.1, July 1981 Printed in U.SA. INTERPRETATION OF PLASMA LUTEINIZING HORMONE ASSAY FOR THE COLLECTION OF MATURE OOCYTES FROM WOMEN: DEFINITION OF A LUTEINIZING HORMONE SURGE-INITIATING RISE JACQUES TESTART, PH.D.** RENE FRYDMAN, M.D.* MARIE CLAUDE FEINSTEIN, PH.D.t ALAIN THEBAULT, M.D.* MARC ROGER, M.D.t ROBERT SCHOLLER, M.D. t Unite INSERM 187, Maternite Antoine Beclere, ClamaTt, France, and Fdndation de Recherche en Horrrwnologie, Fresnes, France Analysis of plasma luteinzing hormone (LH) assays (4 assays per day) permits the discernment of the onset of the physiologic effects of ovulatory release~ This threshold value of LH plasma concentration (LH surge-initiating rise, LH SIR) is determined for each cycle in terms of the average baseline level of the previous day. The chronology of follicle and oocyte maturation after LH SIR is similar to that which follows chorionic gonadotropin (hea) administration: none of the 20 patients, compared with 2 out of 10, had ovulated when laparoscopy was performed 30 to 35 hours and 36 to 38 hours after LH SIR time, respectively; 1 out of 3, 9 out of 14, and 5 out of 5 patients had an in vitro fertilized egg when oocytes were collected 30 to 32, 33 to 35, and 36 to 38 hours after LH SIR time, respectively. Thus the oocyte can be collected 34 to 35 hours after the LH SIR; at this point ovulation has not occurred and the oocyte is capable of being fertilized. Fertil Steril36:50, 1981 The oocyte reaches maturity, i.e., is capable of being fertilized, only in the hours immediately preceding ovulation \ hence the importance of determining accurately the time for collection, especially if the oocyte is intended for in vitro fertilization. At present, the only precise physiologic parameter of oocyte maturity during spontaneous cycles is the detection of gonadotropin-release by the hypophysis. A recognizable peak of folliclestimulating hormone (FSH) occurs simultaneously with the peak of luteinizing hormone (LH)2; however, the FSH surge is less dramatic; hence the more usual recourse to LH detection. Received October 23, 1980; revised and accepted February 10,1981. "'Unite INSERM 187, Maternite Antoine Beclere, 157 rue de la porte de Trivaux, Clamart, France. tfondation de Recherche en Hormonologie, 67 Boulevard Pasteur, Fresnes, France. *Reprint requests: Jacques Testart, Ph.D., Unite INSERM 187, Maternite Antoine Beclere, 157 rue de la porte de Trivaux, Clamart, France. 50 The appearance of LH in the urine could be considerably delayed in comparison with its detection in the peripheral blood.3 Thus only LH assay of blood samples taken with sufficient frequency permits the estimation of the time for mature oocyte collection. The literature remains vague as to the time of ovulation in relation to that of LH-release; ovulation would appear to occur 8 to 9 hours4. 5 or 16 to 17 hours2. 6 after the LH peak. The interval between the beginning of the LH rise and ovulation is even more poorly known: 16 to 56 hours.2, 7-9 The time of ovulation can be more precisely pinpointed with reference to chorionic gonadotropin (heg) administration. Follicular rupture usually occurs about 36 to 42 hours after heg injection. 1O -13 Oocytes for in vitro fertilization are generally collected 29 to 38 hours after heg injection. 12, 14 However, the normal chronology of follicular changes induced by heg can be applied to spontaneous cycles only if a critical blood level of LH capable of producing a

2 Vol. 36, No.1 PLASMA LUTEINIZING HORMONE ASSAY 51 maturational effect on the follicle can be determined. As yet there is no method for interpreting the results of LH assay that enables us to pinpoint the start of preovulatory follicular maturity with an accurate prediction for mature oocyte collection. This study proposes an individualized LH plasma concentration threshold value above which one can suppose the beginning of preovulatory release; the validity of this threshold reposes on two physiologic parameters: the time of ovulation and the fertilizability of the oocyte. MATERIALS AND METHODS Volunteers 25 to 40 years of age agreed to undergo frequent blood sampling prior to endoscopic gynecologic investigation. Most of the patients with bilateral occluded fallopian tubes had been hospitalized for an in vitro fertilization program. Forty-five cycles were studied with regard to the analysis of plasma concentrations of LH on the days preceding preovulatory release. Among these, 30 cycles, studied in 27 patients, allowed the study of the time of ovulation and the fertilizability of the oocyte. In 22 of the 30 cycles no treatment was given; in eight patients the diagnosis of dysovulation led us to adopt clomiphene citrate therapy (Clomid, Merrel-Torraude, 100 mg/day, between days 5 and 9, inclusive, of the cycle). Blood samples were taken four times a day by separate venipuncture at 8 A.M., 1 P.M., 6 P.M., and 11 P.M. from the time at which LH release was considered imminent. The plasma level of LH was determined by rapid radioimmunoassay.3 The intra-assay precision was 6% and 3%, respectively, at mean levels of 2 and 10 miu/ml; the interassay precision was 14% and 9% respectively, at mean levels of 3 and 13 miulml. Thirty to 38 hours after the onset of ovulatory release, as estimated by the method given below, laparoscopy was performed, and revealed the presence of an enlarged follicle or young corpus luteum. The oocytes were collected by aspiration of the follicular contents I5 and the follicular fluid ( ml) was frozen for further investigations. Twenty-six oocytes underwent in vitro fertilization immediately after being collected; each oocyte was placed in the culture medium (B2, Menezo, INRA, API system) containing preincubated spermatozoa. 16 The success of fertilization was appraised by histologic examination after 8 to 57 hours of culture; the presence of two pronuclei and two polar bodies in the undivided ovum, or segmentation into regular and nucleated blastomeres from 35 hours of culture, was considered as the criterion for successful fertilization. RESULTS Analysis of LH Plasma Concentrations Assays Prior to the Preovulatory Release. Mean baselines vary considerably from one patient to another (1 to 6 miulml). During the days preceding LH release, the base level in a given cycle fluctuates considerably. Thus a given assay may indicate an LH concen- TABLE 1. LH Surge-Initiating Rise (LH SIR) Determination (Example: Mrs. T) April 19 April 20 April 21 SA.M. 1 P.M. 6 P.M. 11 P.M. SA.M. 1 P.M. 6 P.M. 11 P.M. SA.M. 1 P.M. LH (miu/mll rise? peak LH SIR value determination Which is the first LH value in the rise? Mean of the four preceding values LH SIR corresponding concentration based on the mean of the four preceding values ( x ) a or LH SIR time determination LH hourly increasing during the rise = 1.5 miu/ml Time elapsed from April 20 at 6 P.M. until the occurrence of LH SIR = 1 hour 40 minutes Time of the LH SIR occurrence 7:40 P.M. ~he value 4.4 does not belong to the rise, since the corresponding LH SIR is superior to this value (4.8 > 4.4).

3 52 TESTART ET AL. July 1981 tration very different from (up to 250%) that found five hours before or after. However, comparison of each result with the mean result of the four preceding assays reduces this difference to less than 80%; it is only shortly before the preovulatory peak that the greatest variations are noted. Definition of the LH Surge-Initiating Rise (LH SIR). If in the present sampling conditions five successive assay results are considered, then any LH level equal to or exceeding 180% of the mean value for the preceding- four assays indicates the onset of the LH surge. The plasma LH concentration corresponding to the rise so defined is termed the LH surge-initiating rise (LH SIR). The time at which the LH SIR appears is evaluated by interpolation from the one sample that precedes and the other one that follows the onset of the LH surge. For this purpose the following assumptions are made: (1) that the LH level at the assay just before that indicating the onset of the LH surge was equal to the mean obtained from the four previous assays (i.e., those of the 24 hours preceding the LH SIR); and (2) that the LH concentration in the plasma began to rise immediately after this assay. Table 1 shows an example of the calculation of the value of the LH SIR and the time at which it appeared. For the 45 studied cycles, the mean LH basal level was 3.95 ± 0.19 miu/ml, and the LH SIR mean value was 7.11 ± 0.35 miu/ml (P < 0.001). Assays Following the LH SIR. Various patterns of changing plasma concentrations may be observed following the onset of LH SIR; among these patterns, the most common (32 cycles) is a regular increase until a peak is reached; this peak is attained with the first rise in four cases. However, the plasma level may stagnate (3 cycles) or even decrease (10 cycles) for five hours before increasing again to the peak. Figure 1A shows an example of a typical release, and Figure 1B shows an "atypical" release, i.e., with an intermediary drop between the LH SIR and the peak. Validation of the LH SIR Tests were carried out with the use of two physiologic phenomena to demonstrate the validity of this interpretation of the assay results. Relationship Between the LH SIR and Ovulation (Table 2). Ovulation is first noted 36 hours after LH SIR (2 out of 10 patients). In the 20 patients who underwent laparoscopy between 30 and 35 hours after the LH SIR the preovulatory follicle was not yet ruptured. The pretreatment with clomiphene had no effect on this result; the eight clomiphene-treated patients had not ovulated between 30 and 38 hours after LH SIR. Relationship Between the LH SIR and the Fertilizability of the Oocyte (Table 3). In nontreated patients, the oocytes were normally fertilized in vitro (10/12) if they were collected at least 33 hours after the LH SIR; two oocytes collected between 30 and 32 hours after the LH SIR were not fertilized. Following clomiphene treatment only 5 out of 12 oocytes were fertilized; but if only one oocyte per patient is taken into account, five patients out of eight presented a fertilized oocyte, which is comparable to the result obtained in nontreated patients. The maximum fertility level in both spontaneous and stimulated cycles is obtained when the oocyte is recovered at least 36 hours after the LH SIR: fertilization was successful in five patients 30 PLASMA LH (rniu/rnl) 20 6 R ",,'. 11pm 8." 1pm 6pm 11pm 8am 1pm 6pm Upm 8am 1pm pm PLASMA B aug. 110m am 1pm 6pm 11pm 8am 1pm TIME OFBlOOD COLLECTION TIME OF BlOOD COLLECTION FIG. 1. a, Typical release with a smooth increase in LH observed in the case of Mrs. R. b, Atypical release with discontinuous rise of LH observed in the case of Mrs. B. In each patient one oocyte was collected 37 hours after the LH surgeinitiating-rise, as indicated. Both oocytes were fertilized in vitro.

4 Vol. 36, No.1 PLASMA LUTEINIZING HORMONE ASSAY 53 TABLE 2. The Relationship Between LH Release (LH SIR) and Ovulation LH SIR--<>varian test delay (hours) Number of patients Ovulation out of five (100%); the success rate is 64.3% (9/14) and 33.3% (113), respectively, if the oocyte is placed in the sperm culture between 33 and 35 hours, or less than 33 hours after the LH SIR. Among the 26 oocytes incubated, five were collease (see RESULTS, Assays Following the LH SIR, page 52); in these five cases, the oocyte collection was carried out 33 to 37 hours after the LH SIR, but only 19 to 27 hours after the second rise oflh towards the peak; four of the five oocytes were fertilized, which is comparable to the result obtained when the LH SIR was established for a cycle presenting a smooth rise in LH. DISCUSSION During the preovulatory period, LH release pulses by the hypophysis occur with a frequency of about one pulse per hour, 17 which explains the fluctuations observed in successive plasma LH concentrations when samples are spaced out several hours apart. It is only by comparing each result with the mean of several previous results that the increase in LH release by the hypophysis can be spotted, i.e., the onset of the LH SIR. The definition of the LH SIR (80% increase relative to the mean level for four preyious assays) is empirical, since what is sought is a physiologic change rather than a statistic of gonadotropic stimulation; the choice of a threshold 10% or 20% higher would only change the timing of the LH SIR by less than one hour, since the rise is always steep. Calculation of the LH SIR could lead to a premature estimate of the LH surge (rather than a delayed estimate), the threshold chosen being just greater than baseline variations; this risk is further increased by the fact that a linear progression of the LH plasma concentration is admitted between the time of the preceding sample and that of the sample indicating the onset of the LH SIR. The demonstration of ovulation in 20% of patients operated on 36 to 38 hours after the LH SIR indicates that the estimated time of the LH surge was not anticipated; this result is similar to that observed after the same delay following hcg administration. A similar situation holds for the fertilizability of the oocyte: the oocyte is regularly fertilized in vitro if it is recovered 36 to 38 hours after the LH SIR; the fertilization rate decreases progressively if the collection is made too soon, probably because of incomplete oocyte maturity. It has recently been reported 9 that 11 out of 15 women ovulated between 24 and 48 hours after the LH rise; the authors concluded that the median time interval from the LH significant rise to ovulation was 32 hours (23.6 to 38.2 hours). In this report, 9 the women were grouped by intervals of eight hours from the hormonal event to operation; the time interpolation in the LH SIR calculation enabled us to expect the occurrence of the LH surge onset a few hours earlier, as compared with the other method. This difference in methods might explain why none of our 17 patients ovulated between 32 and 35 hours after the LH SIR. The determination of the onset of physiologic effects during the gonadotropic release in cycles showing "atypical" LH release poses an acute problem because of the frequency of such cycles (13/45, i.e., 29%). Two successive rises of LH can be identified, separated by a period sometimes exceeding 10 hours; this type of discontinuous release differs from the "biphasic pattern" observed by Thomas et al.,7 the second peak always TABLE 3. Relationship Between the Delay After LH Release (LH SIR) and the In Vitro Fertilization Potential of the Oocyte Treatment No. of oocytes LH SIR--<>ocyte collection interval (hours) Total None Tested (14) Fertilized (10) Clomid Tested 1 (1)a 10 (6) 1 (1) 12 (8) Fertilized 1 (1) 3 (3) 1 (1) 5 (5) Total Tested 3 (3) 18 (14) 5 (5) 26 (22) Fertilized 1 (1) 9 (9) 5 (5) 15 (15) % Patients with one fertilized oocyte anumber of patients.

5 r 54 TESTART ET AL. July 1981 being much greater than the first, and the interval between the peaks less than 24 hours. The results of the in vitro fertilization of these cases (four out of five fertilized oocytes) indicate that tne first increase in the plasma concentration of LH, which corresponds to the LH SIR definition, is the chronologie marker for oocyte maturity, subsequent development of the release having no effect on the phenomenon. This observation also raises the question of the role of massive LH release by the hypophysis during the period following the time when the preovulatory ovarian modifications are induced; it would appear that oocyte maturation induction, like that of follicular maturation, sets in when the plasma level of LH is relatively low. Animal studies show an LH-induced desensitization of the follicle, which is responsible for refractoriness to subsequent gonadotropin administration. 18 The duration of the LH rise, estimated as being the interval between the time of the LH SIR and that of the peak, lasts some 15 hours on average, but varies between 1 and 29 hours in individual cycles. 19 Such variations are without effect on the times of the onset of oocyte maturity or of ovulation, as estimated from the LH sm, since the peak of bloodstream LH is detected at very different phases of follicular maturation from one cycle to another: it may occur 36 or only 7 to 10 hours before ovulation. It is, therefore, only the onset of the LH rise that should be taken into account in the prediction of the most appropriate time for mature oocyte collection. Large variations are observed between the baseline LH levels of different patients; so it is only for a given patient, or even a given cycle, that a certain plasma level can be said no longer to correspond to the tonic release, but to mark preovulatory release. Our results show that the chronology of preovulatory events after the LH SIR is similar to that which follows the injection of an adequate dose of hcg. Reference to the LH SIR provides adequate precision concerning follicular maturation to ensure the in vitro fertilization of oocytes recovered during spontaneous cycles, providing that a rapid blood LH assay method is available. Acknowledgment. Special thanks are extended to Bruno Lassalle for excellent technical assistance. REFERENCES 1. Edwards RG: Studies on human conception. Am J Obstet GynecoI117:587, Yussman MA, Taymor ML, Miyata J, Pheteplace C: Serum levels of follicle-stimulating hormone, luteinizing hormone, and plasma progestins correlated with human ovulation. Fertil Steril 21:119, Roger M, Grenier J, Houlbert C, Castanier M, Feinstein MC, Scholler R: Rapid radioimmunoassays of plasma LH and estradiol-1713 for the prediction of ovulation. J Steroid Biochem 12:403, Lunenfeld B, Ben-Aderet N, Ben-Michael R, Grunstein S, Kraiem Z, Potashnik G, Rofeh C, Shalit A, Tikotsky DT: Temporal relationship between hormonal profile and ovarian morphology during the periovulatory period in human. In Endocrinology of the Ovary, Edited by R Scholler. Paris, Sepe, 1978, p Pauerstein CJ, Eddy CA, Croxatto HD, Hess R, Siler Khodr TM, Croxatto HB: Temporal relationship of estrogen, progesterone and luteinizing hormone levels to ovulation in women and infrahuman primates. Am J Obstet Gynecol 130:876, Croxatto HD, Ortiz MA, Croxatto HB: Correlation between histologic dating of human corpus luteum and the luteinizing hormone peak-biopsy interval. Am J Obstet GynecoI136:667, Thomas K, Walckiers R, Ferin J: Biphasic pattern oflh mid-cycle discharge. J Clin Endocrinol Metab 30:269, Ben-Aderet N, Potashnik G, Lunenfeld B, Kraiem Z, Grunstein S, Shalit A: Correlation of hormonal profile and ovarian morphologic features during the periovulatory period in humans. Fertil Steril 28:361, World Health Organization Task Force: Temporal relationships between ovulation and defined changes in the concentration of plasma estradiol-1713, luteinizing hormone, follicle-stimulating hormone, and progesterone. I. Probit Analysis. Am J Obstet Gynecol 15:383, Edwards RG, Steptoe PC: Control of human ovulation, fertilization, and implantation. Proc Roy Soc Med 67:932, Jagiello G, Karnicki J, Ryan R: Superovulation with pituitary gonadotrophins. Lancet :178, Steptoe PC, Edwards RG: Laparoscopic recovery of preovulatory human oocytes after priming the ovaries with gonadotrophins. Lancet 1:683, Testart J, Thebault A, Frydman R: Oocyte maturity and in vitro fertilization in human. World Congress on Embryo Transfer, In Vitro Fertilization and Instrumental Insemination, Kiel (W. G.). Arch Androl 5:74, Lopata A, Johnston IWH, Leeton JF, Muchnicki D, Talbot J, Wood C: Collection of human oocytes at laparoscopy and laparotomy. Fertil Steril 25:1030, Frydman R, Feinstein MC, Testart J, Thebault A: Recovery of human preovulatory oocyte by LH plasma rapid radioimmunoassay method. World Congress on Embryo Transfer, In Vitro Fertilization and Instrumental Insemination, Kiel (W. G.). Arch Androl 5:85, Testart J, Thebault A, Frydman R: La fecondation in vitro humaine: nos premiers resultats. Gynecol Obstet BioI Reprod 9:139, Yen SSC, Tsai CC, Naftolin F, Vandenberg G, Ajabor L: Pulsatile patterns of gonadotrophin release in subjects with and without ovarian function. J Clin Endocrinol Metab 34:671, Lindner HR, Amsterdam A, Solomon Y, Tsafriri A, Nimrod A, Lamprecht SA, Zor U, Koch Y: Intraovarian factors in ovulation: determinants offollicular response to gonadotrophins. JReprod Fertil 51:215, Testart J, Frydman R: Unpublished data