Evaluation of diagnostic ultrasound as a parameter of follicular development in an in vitro fertilization program

Scientific Articles FERTILITY AND STERILITY Copyright 1983 The American Fertility Society

Vol. 39, No.4, April 1983 Printed in U.8A.

Evaluation of diagnostic ultrasound as a parameter of follicular development in an in vitro fertilization program

Beresford Buttery, M.B., B.S., F.RA.C.O.G., D.D.U.* Alan Trounson, Ph.D., M.Sc. Rex McMaster, B.Med.Sci. Carl Wood, M.B., B.S., F.RC.O.G., F.RA.C.O.G., F.RC.S. Department of Obstetrics and Gynaecology, Monash University, Queen Victoria Medical Centre, Melbourne, Victoria, Australia

The ultrasound results in 141 patients through 203 cycles stimulated with clomiphene citrate prior to oocyte collection and in vitro fertilization are analyzed. A wide range in the size and number of follicles was seen. Bivariate analysis of the size of the largest follicle, compared with the time of the spontaneous luteinizing hormone (LH) surge, has shown a highly significant correlation (r = 0.720; P < 0.001) between these two variables, but the accuracy of prediction of the LH surge in a specific cycle, as expressed by the standard error of estimate, was 34.3 hours. As an isolated observation, ultrasound results are no better than the menstrual history in predicting the time of the LH surge in stimulated cycles. Multiparameter assessment of follicular development is required. Fertil Steril39:458, 1983

Since the initial report of the ultrasonic visualization of the ovaries by Kratochwil et al. l and the subsequent studies of developing follicles in the normal and stimulated menstrual cycles, diagnostic ultrasound has been found to be of value in an in vitro fertilization (IVF) program. 2 Although early attempts at IVF and embryo transfer (ET)3 were made following ovarian stimulation, the first successful pregnancy was attained following a spontaneous ovulatory cycle, 4 and the assumption was made that optimal results would be obtained from this regimen of management. Despite the initial failures, it has now been shown that ovarian stimulation im-

proves the success rate of IVF-ET and has many advantages when compared with programs using the natural ovulatory cycle. 5 , 6 Although several follicles may be present in the spontaneous cycle, usually only one follicle increases in size beyond 1.4 cm in diameter. This follicle is most likely to ovulate, and it has been shown that the size of the largest follicle is closely correlated with peripheral blood estrogen levels. 7 By comparison, in stimulated cycles not only are multiple follicles present, but there is a wide variation in their size. 8 In this study, we analyzed the results of 362 ultrasound examinations in 141 patients through 203 stimulated cycles to evaluate diagnostic ultrasound as an index of follicular development.

Received September 20, 1982; revised and accepted December 14, 1982. *Reprint requests: Dr. Beresford Buttery, Department of Obstetrics and Gynaecology, Queen Victoria Medical Centre, Melbourne, Victoria 3000, Australia.

MATERIALS AND METHODS

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In this series all patients were given 150 mg clomiphene citrate (Clomid, S. Merrel Pty. Ltd., Sydney, Australia) daily from days 5 to 9 of the Fertility and Sterility

cycle. Plasma 17~-estradiol (E 2) levels were monitored daily the the use of a rapid radioimmunoassay (RIA).9 An ultrasound examination was performed on day 10, and if the diameter of the largest follicle was 1. 7 cm or more, the patient was admitted to the hospital, and urinary luteinizing hormone (LH) levels were estimated every 3 hours with the use of a hemagglutination assay (Higonavis, Mochida Pharmaceutical Company, Tokyo, Japan) or RIA.10 Laparoscopy was performed 25 to 27 hours after the onset of the spontaneous LH surge. 5 If the largest follicle was less than 1.7 cm in diameter, further ultrasound examinations were performed at 1- or 2-day intervals, depending on follicular size, until the largest follicle measured 1.7 cm in diameter. In cycles that were controlled, ovulation was induced by administration of 4000 IU of human chorionic gonadotropin (hCG) (Pregnyl, Organon, Australia Pty. Ltd., Clayton, Australia). The time of the injection was based on various criteria,9, 11 including estrogen levels (400 to 500 pg/ml E2 for each large follicle present), the presence of ovulatory cervical mucus, and the absence of a spontaneous LH surge. ULTRASOUND EXAMINATION

The patients were scanned with Emisonic 4200E static B-scan equipment (Nuclear Enterprises Ltd., Edinburgh, Scotland). All patients were examined by one person (B. B.) to avoid observer error. Early in this series, patients were scanned preoperatively with an ADR2130 linear phased array real-time scanner (ADR Ultrasound Corporation, Tempe, Arizona). However, this regimen was abandoned, because results were often inaccurate. In one case no follicles were seen prior to laparoscopy; yet a successful ovum collection, IVF, ET, pregnancy, and delivery of a healthy infant occurred. Where possible, all follicles were measured in three planes, and an average follicular diameter was determined. The internal diameter was measured directly from the echogram with the use of the calibration markers. The system velocity was 1540 mlsecond. When multiple follicles are present, they may be conical in shape (Fig. 1), and measurement in three planes is difficult. As the position of the ovary may vary, depending on the degree of fullness of the bladder, when multiple follicles (Fig. 2) are present, serial examinations to assess the growth rate of a specific follicle have been found to be unreliable. Vol. 39, No.4, April 1983

We estimated the number of follicles present and used the results to assess the plasma estrogen levels. 11 In some cases, because only one ovary was accessible to laparoscopic aspiration, the distribution as well as the number and size of follicles in each ovary was determined prior to ovum collection. The length of the uterine cavity was measured because this estimate is necessary to correctly position the embryo at the time of ET12 and to reduce the chance of tubal pregnancy.13 A general assessment of the pelvis and lower abdomen is made to detect and evaluate any associated pathology. ANALYSIS OF RESULTS

The time of the spontaneous LH surge has been taken as the basis for analysis because this parameter is currently the most accurate method of predicting the time of ovulation, 14 when it is most likely that at least one mature follicle will be present. We investigated the relationship between follicular diameter as determined by ultrasound and the time of the LH surge with the use of regression analysis. The relationship between the day of the cycle (as determined from the first day of menstruation) and the LH surge was similarly investigated.

Figure 1 Longitudinal echogram of an ovary illustrating the triangular appearance of a conical-shaped follicle. A I-em calibration (C) is shown. Buttery et al.

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Figure 2 Transverse echogram in which three large follicles (FJ are seen. The uterus (U) is shown. Calibration markings (C) are at I-cm intervals.

Reported are the regression coefficient, which is a measure of the predictive relationship between the two variables, and the standard error of estimate (SEE), which is a measure of prediction accuracy. The latter statistic is determined by testing the accuracy of the regression equation with the use of the raw data.

proved when other parameters, such as the average diameter of all follicles seen or the average diameter of all follicles 1.5 cm or greater, were analyzed. The SEE values in these instances were 34.6 and 35.8 hours, respectively. In this same group, the SEE for prediction of the spontaneous LH surge from the date of the last menstrual period was 35.4 hours. As an isolated observation, the ultrasound results are no better than the menstrual history in predicting the time of the spontaneous LH surge in stimulated cycles. The average size of the largest follicle at the time of the LH surge is 1.9 cm, but the diameter may range between 1.5 and 2.3 cm. With the exception of one cycle, the LH surge did not occur within 24 hours if the largest follicle was less than 1.5 cm in diameter. The average growth rate of the entire follicle population (836 follicles) was 1.4 mmJday whereas the average growth rate of the largest follicle was 1.6 min/day. The results of treatment are summarized in Table 1. The percentages of cycles in which 00cytes were obtained, in which embryos developed, and in which embryos were transferred were similar. The clinical details of the nine pregnancies are reported elsewhere. 6 In this particular group of patients, there was an indication that the pregnancy rate was higher when a spontaneous 30

RESULTS In this series there were 67 cycles in which a spontaneous LH surge occurred, and in 136 cycles ovulation was controlled with heG. The size of the follicles observed, compared with the time of the last menstrual period in 50 cycles randomly selected from this series, is shown in Figure 3; and the number of follicles in all cycles (Fig. 4) illustrates the wide range of size and number of follicles that develop after ovarian stimulation. There were no cases of hyperstimulation. Bivariate analysis of the size ofthe largest follicle, compared with the time of the spontaneous LH surge in the 62 cycles in which laparoscopy was performed, has confirmed a highly significant correlation (r = 0.720; P < 0.001) between the two variables (Fig. 5). However, the accuracy of prediction of the LH surge from the ultrasound results in an individual case, as expressed by the SEE, was 34.3 hours. This accuracy was not im460

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LH surge occurred, although the numbers are small. A complete analysis of all our data is currently being made. DISCUSSION

In contrast to infertility programs employing ovarian stimulation followed by in vivo fertilization, where there is a significant risk of uncontrolled polypregnancy, IVF programs carry no such hazard because the number of embryos transferred is controlled. In fact, our aim is to produce multiple mature follicles in order to improve the pregnancy rate following IVF-ETI5 and reduce the number of laparoscopic ovum collections required for success. In this study we have found that in stimulated cycles the ultrasonic measurement of the size of the largest follicle or the average size of all follicles present or the average size of all follicles equal to or greater than 1.5 cm in diameter is no more accurate than the menstrual history in predicting the time of the LH surge. Diagnostic ultrasound is an indirect method of assessment of · follicular maturity; and when factors such as observer error, technical difficulties due to associated pelvic pathology, and distortion of echograms due to anterior abdominal wall scars are also taken into account, even in optimal circumstances interpretation of results in an individual case can only be an approximation of the status quo. To assume that the size of the largest follicle is an accurate index of follicular development may result in a less than optimal yield of mature 00cytes. Although successful IVF pregnancies have Vol. 39, No.4, April 1983

been reported with the use of ultrasound and cervical mucus changes to assess follicular development, 5 the success rate is significantly improved when estrogen secretion is the primary monitor and ultrasound, together with cervical mucus changes, is used to assist the interpretation of estrogen patterns. 11 We are currently developing a regression equation computed following analysis of all parameters from successful pregnancies in order to improve the accuracy of prediction of follicular development and time of hCG administration. In stimulated cycles the largest follicle may not necessarily be a mature follicle. It has been shown that oocytes that give rise to successful pregnancies are obtained from follicles that contain a greater concentration of E2 and that the largest follicle is not necessarily the most estrogenic. 16 Moreover, mature oocytes have been obtained from relatively small follicles. Successful IVF pregnancies have occurred following collection of oocytes from follicles as small as 1.6 cm in diameter, and apparently normal embryos have developed from 39% of ova obtained from follicles with volumes between 1 and 2 ml. 11 Recent studies in the primate suggest that the time of administration of hCG is critical for the 35 hrs to LH peak ..

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Table 1. Comparison of the Results of Treatment in Stimulated and Controlled Cycles Spontaneous Administration LH surge of heG Significance (stimulated (controlled
No. of cycles No. of laparoscopies No ovum collected-not ovulated No ovum collected-ovulated No fertilization Abnormal development Embryo(s) transferred Successful pregnancies

136 105

67 62 4 (6.5%)a

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12 (11.4%) 3 (2.9%)

0 17 (27.4%) 3 (4.8%)

26 (24.8%) 6 (5.7%)

NS NS

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58 (55.2%)

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subsequent release of oocytes capable of undergoing fertilization. 17 An ovulation rate as high as 92% has been reported when ultrasound results were used to determine the time of administration of heG; however, up to six treatment cycles were required for a pregnancy rate of 67%.18 We consider that administration of heG for an IVF program based on ultrasound results alone 19 cannot be justified. There is no "ideal" size when it can be assumed that a follicle is mature. Other authors8, 20, 21 have also reported similar conclusions. Nevertheless, estimation of the size of follicles present is still of value. If the follicles are extremely small or if there is evidence of hyperstimulation, the observations can be used to decide whether a further ultrasonic examination is warranted for the assessment of follicular growth or whether the treatment cycle should be abandoned. Provided follicular size is within normal limits, the diameter of the largest follicle may be used to determine when the patient should be admitted to the hospital for more intensive monitoring of follicular development measured by daily estrogen levels and the spontaneous LH surge estimated by urine samples taken every 3 hours. The ultrasonic assessment of follicular growth in stimulated cycles is particularly difficult. The largest follicle on day x may not necessarily be the largest on day x + 2, because individual follicles may show different growth rates 22 and the largest follicle may be atretic. 16 The growth rate may be between 1 and 4 mmlday,23 and in this 462

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study the average growth rate of the largest follicle, as expressed by the regression equation, was 1.6 mmlday. This general trend in the growth of follicles has been found to be of value in the interpretation of results in patients who require serial ultrasound examinations prior to admission to the hospital. The cumulus ovarii may be visualized with the use of ultrasound; however, this feature was only occasionally seen in this series, because most scans were performed prior to the LH surge. A recent study24 comparing ultrasonic details with the histologic appearance of follicles indicates the potential of ultrasound in the functional assessment of follicles. Further studies are warranted for the critical evaluation of the ultrasonic appearance of the stimulated follicle in relation to atresia and maturity of the oocyte. Evaluation of the number and distribution of follicles is necessary for the adequate interpretation of estrogen levelsl l and the rational management of patients when only one ovary is accessible to laparoscopy. Even if the largest follicle is in the inaccessible ovary, the treatment cycle does not have to be abandoned, provided that there are several follicles in the contralateral ovary within the normal range. As yet, we have not attempted ultrasonically guided percutaneous aspiration of oocytes,25 but the technique could be of value when no follicles are present in the accessible ovary or when both ovaries are inaccessible to laparoscopy. We have found that the ultrasound results in a particular cycle are of value for interpretation of the ovarian response to stimulation, timing of admission to the hospital for the intensive monitoring of follicular development and administration of heG, assessment of the accessibility of follicles for laparoscopic ovum collection, evaluation of the uterine position and dimensions for ET, and the diagnosis of associated uterine or pelvic disease. We believe that the monitoring of follicular development by ultrasound alone is inadequate for an IVF program. REFERENCES 1. Kratochwil A, Urban G, Friedrech F: Ultrasonic tomography of the ovaries. Ann Chir Gynaecol (continues Ann Chir Gynaecol Fenniae) 61:211, 1972 2. de Crespigny LJCh, O'Herlihy C, Hoult IJ, Robinson HP: Ultrasound in an in vitro fertilization program. Fertil Steril 35:25, 1981

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3. Steptoe PC, Edwards RG: Laparoscopic recovery of preovulatory oocytes after priming ovaries with gonadotrophins. Lancet 1:683, 1970 4. Edwards RG, Steptoe PC, Purdy JM: Establishing fullterm pregnancies using cleaving embryos grown in vitro. Br J Obstet Gynaecol 87:737, 1980 5. Trounson AO, Leeton JF, Wood C, Webb J, Wood J: Pregnancies in humans by fertilization in vitro and embryo transfer in the controlled ovulatory cycle. Science 212:681, 1981 6. Wood C, Trounson A, Leeton J, Talbot JMcK, Buttery B, Webb J, Wood J, Jessup D: A clinical assessment of nine pregnancies obtained by in vitro fertilization and embryo transfer. Fertil Steril 35:502, 1981 7. Kerin JF, Edmonds DK, Warnes GM, Cox LW, Seamark RF, Matthews CD, Young GB, Baird DT: Morphological and functional relations of Graafian follicle growth to ovulation in women using ultrasonic, laparoscopic and biochemical measurements. Br J Obstet Gynaecol 88:81, 1981 8. Seibel MM, McArdle CR, Thompson IE, Berger MJ, Taymor ML: The role of ultrasound in ovulation induction: a critical appraisal. Fertil Steril 36:573, 1981 9. Trounson AO, Leeton JF, Wood C: In vitro fertilization and embryo transfer in the human. In Follicular Maturation and Ovulation, Edited by R Rolland, EV Van Hall, SG Hillier, KP McNatty, J Schoemaker. Amsterdam, Excerpta Medica, 1982, p 313 10. Trounson A, Herrios M, Burger H, Clarke I: The precise detection of ovulation using a rapid radioimmunoassay of urinary LH. Proc Endocrine Soc Aust 23:72, 1980 11. Trounson AO: In vitro fertilization. In Current Topics in Experimental Endocrinology: Pregnancy and Parturition, Vol. 5, Edited by L Martini, V James. New York, Academic Press, 1983, p 44 12. Leeton J, Trounson A, Jessup D, Wood C: The technique for human embryo transfer. Fertil Steril 38:156, 1982 13. Steptoe PC, Edwards RG: Reimplantation of a human embryo with subsequent tubal pregnancy. Lancet 1:880, 1976 14. Collins WP, Collins PO, Kilpatrick MJ, Manning PA, Pike JM, Tyler JPP: The concentration of urinary estrone-3-glucuronide, LH and pregnanediol-3o:-glucuronide as indices of ovarian function. Acta Endocrinol (Copenh) 90:336, 1979

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15. Trounson A: Current perspectives of in vitro fertilization and embryo transfer. Clin Reprod Fertil 1:55, 1982 16. Carson RS, Trounson AO, Findlay JK: Successful fertilization of human oocytes in vitro: concentration of estradiol-17j3, progesterone and androstenedione in the antral fluid of donor follicles. J Clin Endocrinol Metab 55:798, 1982 17. Williams RF, Hodgen GD: Disparate effects of human chorionic gonadotropin during the late follicular phase in monkeys: normal ovulation, follicular atresia, ovarian acyclicity, and hypersecretion of follicle-stimulating hormone. Fertil Steril 33:64, 1980 18. O'Herlihy C, Pepperell RJ, Robinson HP: Ultrasound timing of human chorionic gonadotropin administration in clomiphene stimulated cycles. Obstet Gynecol 59:40, 1982 19. Hoult IJ, de Crespigny LCh, O'Herlihy C, Speirs AL, Lopata A, Kellow G, Johnston I, Robinson HP: Ultrasound control of clomiphene/human chorionic gonadotropin stimulated cycles for oocyte recovery and in vitro fertilization. Fertil Steril 36:316, 1981 20. Salat-Baroux J, Scemama-Kestenberg H, Levy G, Baillet C, Couturier JY: Experience with 110 cases of ovulation inductions, monitored in the plasma and by means of ultrasound: research to establish a criterion of follicular maturation. Acta Eur Fertil 12:59; 1981 21. Queenan JT, O'Brien GD, Bains LM, Simpson J, Collins WP, Campbell S: Ultrasound scanning of ovaries to detect ovulation in women. Fertil Steril 34:99, 1980 22. Cabau A, Bessis R: Monitoring of ovulation induction with human menopausal gonadotropin and human chorionic gonadotropin by ultrasound. Fertil Steril 36:178, 1981 23. YlOstalo P, Ronnberg L, Jouppila P: Measurement of the ovarian follicle by ultrasound in ovulation induction. Fertil Steril 31:651, 1979 24. Bomsel-Helmreich 0: Preovulatory human oocyte and its microenvironment. In Fertilization of the Human Egg in Vitro: Biological Basis and Clinical Applications, Edited by HM Beier, HR Linder. Berlin, Springer-Verlag, 1982. In press 25. Lenz S, Lauritsen JG, Kjellow M: Collection of human oocytes for in vitro fertilization by ultrasonically guided follicular puncture. Lancet 1:1163, 1981

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