Concepts in human in vitro fertilization and embryo transfer

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1 FERTLTY AND STERLTY Copyright Q 1983 The American Fertility Society Vol. 40, No.3, September 1983 Printed in U.8A. Concepts in human in vitro fertilization and embryo transfer Alexander Lopata, M.B., B.S., Ph.D. University of Melbourne and Reproductive Biology Unit, Royal Women's Hospital, Melbourne, Victoria, Australia ed lid ke ral ~a; ne :al ~a. ay lie le in ~or ng be s. 'e.,sl ge ng lie ed ng he or,el lur sal nd ng be s. he ial.01 )/e ng ns en ns 3X, 01,,lie 83 At the end of 1982, there were about 20 clinics in the world possessing the expertise for producing laboratory conceptuses which, when placed in the uterus of infertile women, were capable of progressing to term pregnancies. The combined work of about ten such clinics has resulted in the birth of over 100 infants by the beginning of the current year. t may be confidently predicted that before the end of the next 12 months there will be about 50 successful clinics, and over 300 children will have been born to infertile couples as a result of in vitro fertilization (lvf) and embryo transfer (ET). The clinics achieving pregnancies will be able to evaluate the ovarian stimulation protocols, the in vitro culture conditions, and the ET techniques for attaining the highest success rates. Some of this information has already been presented at specialized meetings and will be published in books. 1, 2 n addition, the combined results of the six successful clinics in Australia have recently been analyzed in book form. 3 Because we do not have prepublication access to these tomes, a discussion of the comparative data contained therein will need to be tackled by a future reviewer. The rapid increase of VF clinics throughout the world has resulted in the rapid growth of information in this field. Because publication is a relatively slow method of disseminating such information, much time is wasted in duplicating work and in adopting procedures already shown to yield poor results. The establishment of an nternational Register for storing, correlating, and disseminating human VF-ET data was proposed at a meeting in Bourn Hall. 4 Although there was general agreement about the need for an VF Register, no decisions were made about its location, its operation, and the sort of data it should Vol. 40, No.3, September 1983 attract. There is now an urgent need to take further steps to implement an nternational Register. n view of the paucity of readily available information of sufficient magnitude for comparative analysis, the present review will largely evaluate the outcome of 722 laparoscopies carried out for VF-ET at the Royal Women's Hospital from 1980 through The analysis will deal exclusively with stimulated menstrual cycles to evaluate the ovarian stimulation and monitoring protocols, the in vitro culture procedures, and the ET methodology. The work will be described in two parts, based on the clinical procedure used for monitoring fol~ licular maturation. The first part covers the period 1980 and 1981, when ultrasound was used as the sole method for assessing the state of the leading follicle. The second part spans the whole of 1982, when monitoring of follicular growth and maturation was assessed by means of measurement of daily plasma estradiol (Ez) levels in combination with pelvic ultrasonography. PART 1: STMULATED CYCLES N THE 1980 AND 1981 PROGRAM The selection of patients for VF-ET has been discussed in detail. 5-7 Table 1 is based on 1981 data and shows the main infertility factors in a representative group of patients treated in our program. All women were treated with clomiphene citrate (CC) and human chorionic gonadotropin (hcg) according to the following protocol. (1) CC, 100 mg daily, was administered from days 5 to 9 of the menstrual cycle. (2) Daily ultrasonography of the ovaries was carried out from day 10 of the cycle. Lopata Concepts in VF -ET 289

2 Table 1. The Main nfertility Groups Treated by NF-ET in 1981 Total patients Tubal disease Abnormal semen Endometriosis mmunologic (female) Unexplained % 14.8% 8.7% 8.3% 14.0% (3) The patient was admitted to the hospital when the average diameter of the leading follicle, measured in three planes, was ;;;. 18 mm. (4) A blood sample was taken for rapid luteinizing hormone (LH) assay, and then 5000 U ofhcg was administered intramuscularly. (5) f the LH level was basal, laparoscopy for follicular aspiration was performed - 36 hours after the hcg injection. f the LH level was elevated, indicating that the endogenous LH surge had commenced, the patient was discharged. Modified Ham's F-10 medium8 containing 10 vol% of the patient's inactivated serum was used for in vitro insemination. The ph of this medium was 7.45 to 7.50 until mid-1981, when it was subsequently lowered to 7.30 to The ph of the embryo culture medium was maintained at 7.30 to 7.35 throughout the period. The embryo growth medium contained 15 vol% of maternal serum. The osmolarity of both media was adjusted to mosm/kg. Fresh medium was prepared each week, and all cultures were carried out in 1-ml aliquots contained in loosely capped Falcon tubes (#2003, Falcon Plastics, Oxnard, CA) in a humidified atmosphere of 5% CO2 + 5% O2 + 90% N2 at 37 C. n some cases the K+ level in the Ham's F-lO was raised to - 10 mm, and the medium was supplemented with 0.5 mm MgS0 4 n 1980 and for the first 5 months of 1981, the aspirated eggs were transferred into culture medium and inseminated immediately with 1 x 10 6 motile spermatozoa per milliliter of medium. The preparation and preincubation of spermatozoa has been described previously. 5-8 After 8 to 24 hours of insemination culture, the eggs were transferred into the embryo culture medium. At 36 to 48 hours after insemination, the corona cells were removed to evaluate embryo growth. Cleaving embryos were cultured for a further 12 to 36 hours; and then one (but occasionally two) embryo was transferred into the ovum donor's uterine cavity. At this stage of the program, all embryos inserted into the uterus were at about the 8-cell stage of development. 290 Lopata Concepts in VF-ET nitially, nylon catheters were used for ET, but subsequently fine Teflon catheters were adopted. The influence of various catheter types on the outcome of ET has been discussed by various authors.5, 9-12 n the last 6 months of 1981, several changes were introduced into the program: (1) The hcg injection was delayed until the average diameter of the leading follicle was ;;;. 20 mm. (2) Preovulatory eggs were not inseminated immediately after their aspiration. nstead, the eggs were incubated for 3 to 6 hours before sperm were added. (3) Embryos were transferred into the uterus at earlier stages of growth, ranging from the 2-cell to the 8-cell stage of development. However, as in the first half of the year, only one or two embryos were implanted. RESULTS A comparison of the results obtained in 1980 and 1981 is presented in Table 2. Although the ovarian stimulation protocol and the procedure for follicular aspiration were similar in the 2 years, a slightly. higher average number of preovulatory oocytes was obtained in The fertilization rate increased significantly from 55.3% in 1980 to 66.3% in 1981 (X 2 = 5.64, P < 0.02). Concomitantly there was a highly significant increase in the proportion of embryos obtained from inseminated eggs (X 2 = 6.92, P < 0.01) and in the proportion of patients having ET relative to the number of laparoscopies performed in each year (X2 = 1l.85, P < 0.001). n a subsequent section, the above results will be evaluated in terms of the effects of immediate and delayed insemination of Table '2. Comparison of i1gg Recovery, Fertilization, ET, and the Outcome of Pregnancies in 1980 and Laparoscopies 93 Preovulatory fol- 211 (2.3/lapa) licles Eggs recovered 157 (1.7lap) Eggs inseminated 152 Eggs fertilized 84 (55.3% insemb) Embryos transferred.45 Transfer patients 35 (37.6% lap) Term pregnancies C 1 Abortions 2 alaparoscopies. bnseminated. cfive normal infants were delivered (2.7lap) 512 (2.0lap) (66.3% insem) (59.1% lap) 3 (1 twin) 14 Fertility and Sterility

3 A60 A:j STMULATED CYCLES & MMEDATE NSEMNATON [Embryo development in relation to follicle size Goon" Embryos transferred 8: w g ~ :> ~ ~.,. j1,g ;f.,. 40 B n,51 n,9 n,11 n 81 Size of preovulatory tollie" hcg,dlam.1 S-2 0cml Total follicl OLL~~~~~~~~~~~~~ n M ~ ~ ro H H H H W H Follicle Diameter - cm Figure 1 The distribution of follicle sizes, and the performance of eggs derived from them, in patients treated with CC, 100 mg daily, from days 5 to 9 of the menstrual cycle. n these patients, hcg was administered when the leading follicle attained a diameter of;;;. 18 mm, and oocytes were inseminated immediately after their aspiration. The lower histogram shows the distribution of follicle sizes as a percentage of the total follicle population. The upper histogram indicates the percentage of follicles of each size that yielded eggs that became fertilized and produced normally cleaving embryos. The closed circles represent embryos that produced pregnancies. preovulatory eggs. The pregnancies achieved and their outcome are indicated in Table 2, but statistical analysis is not warranted in view of the low numbers. When the timing of hcg injection was based on a leading follicle attaining ~ 18 mm in diameter, the population of follicle sizes found at laparoscopy is shown in Figure 1. As may be seen from the lower histogram, - 25% of aspirated follicles were > 22 mm in diameter following this protocol. f hcg was given when the average diameter of the leading follicle was ~ 20 mm, - 42% of follicles were> 22 mm at laparoscopy (Fig. 2, lower histogram). There was a statistically significant difference between the two populations of follicle sizes. The upper histogram in Figures 1 and 2 compares the ability of follicles of various sizes to produce eggs that will fertilize and give rise to embryos suitable for transfer into the uterus. The results presented in these histograms depend on the oocyte recovery rate, fertilization rate, and cleavage success rate in relation to follicle sizes. As may be seen by comparing Figures 1 and 2, the delayed insemination of oocytes has improved the percentage yield of embryos for most follicle sizes. Another parameter we investigated was whether following ovarian stimulation there was a difference in the fertilization rate of eggs derived from leading follicles (largest diameter) compared with eggs derived from secondary follicles. The results are summarized in Table 3. When oocytes were inseminated immediately after their recovery, the fertilization rate of eggs from leading follicles was - 15% higher than that of eggs from secondary follicles (Table 3); this difference was not statistically significant for the groups analyzed (X 2 = 2.76,0.5 < P < 0.1). When insemination was delayed for 3 to 6 hours, a fertilization rate - 70% was obtained for eggs derived from leading and secondary follicles (Table 3). Similarly, there was no significant difference in the fertilization rate for eggs from leading follicles irrespective of whether insemination was immediate or delayed. On the other hand, when eggs ob- STMULATED C'rtLES & DELAYBl NSEMNATON Embryo ct.velopment n... on to tollid... neembrjqa t,... Figure 2 The distribution of follicle sizes, and the performance of eggs derived from them, in patients treated with CC, 100 mg daily, from days 5 to 9 of the menstrual cycle. n these patients, hcg was administered when the leading follicle attained a diameter of;;;. 20 mm, and oocytes were incubated for 3 to 6 hours before they were inseminated. The lower histogram shows the distribution of follicle sizes as'a percentage of the total follicle population. The upper histogram indicates the percentage of follicles of each size that yielded eggs that became fertilized and produced normally cleaving embryos. The closed circles represent embryos that produced pregnancies, and the open circles represent embryos associated with pregnancies following multiple ET. 3 4 Vol. 40, No.3, September 1983 Lopata Concepts. in VF-ET 291

4 Table 3. Fertilization of Preovulatory Eggs Recovered from Dominant and Secondary Follicles Following mmediate nsemination and Delayed (3 to 6 Hours) nsemination Dominant follicles mmediate insemination Total eggs 61 Fertilized 38 Fertilization 62.3% rate Delayed insemination Total eggs 151 Fertilized 109 Fertilization 72.2% rate anot significant. NS Secondary follicles % % 14.27, P < mined from secondary follicles were compared, it became evident that preincubation prior to insemination significantly increased their fertilization rate (X 2 = 14.27, P < 0.001). This suggests that maturation of eggs derived from subordinate follicles during preincubation largely accounts for their enhanced fertilizability. Because all preovulatory eggs were inseminated immediately after their retrieval during 1980 and the first half of 1981 and all eggs were cultured up to 6 hours before sperm were added in the second half of 1981, it is possible to analyze the overall outcome of delayed insemination. Tables 4 to 6 compare the fertilization, cleavage, and blastocyst implantation success rates following immediate and delayed insemination ofpreovulatory eggs. The highly significant increase in the fertilization rate is the most dramatic effect of delayed insemination. However, after fertilization has occurred, there is no significant difference in the onset of cleavage between the two groups of eggs (Table 5). Nevertheless, a further difference between the two groups of eggs appears to be the greater capacity of preincubated eggs to produce embryos which progress to implanting blastocysts (Table 6). NS PART 2: THE OUTCOME OF FOUR PROTOCOLS FOR OVARAN STMULATON n 1982 daily measurements of plasma E2 were introduced and used in conjunction with ultrasonography to assess follicular growth and maturation. After admission to the hospital all patients collected urine specimens every 3 hours until the. pre-hcg blood sample was taken for measuring 292 Lopata Concepts in VF-ET the endogenous LH level. f the level was elevated, the LH content of the urine samples was measured, using Hi-gonavis (Mochida Pharmaceuticals, Tokyo, Japan) so that the time of onset of the LH surge could be determined. n patients with a spontaneous LH rise, laparoscopy for oocyte collection was carried out 24 to 27 hours after the onset of the urinary LH surge. n patients with a basal LH level, follicular aspiration was performed 36 to 37 hours after the intramuscular injection of 5000 U of hcg. The following ovarian stimulation protocols were evaluated in 1982: Protocol (1) CC, 100 mg or 150 mg daily, was administered from days 5 to 9 of the menstrual cycle. (2) Ultrasonographic assessment of follicular growth was begun on day 9 of the cycle and repeated on alternate days. (3) The patient was admitted to the hospital when the ratio of plasma E2 to leading follicles was pg/ml per follicle. (4) f the endogenous LH was basal, hcg was administered when plasma E2 was over 500 pg/ml for each follicle> 14 mm in diameter. Alternatively, hcg can be given when the leading follicle is > 20 mm in diameter. Protocol CC, 100 mg or 150 mg daily, was administered starting from day 3 of the menstrual cycle. n this protocol, CC was usually administered for longer than the conventional period to stimulate the growth of a larger number of follicles. As a general rule, CC treatment was continued until the leading follicle was > 14 mm in diameter. Otherwise, this protocol was similar to protocol. Protocol CC was used in combination with human menopausal gonadotropin (hmg) in protocol. Table 4. The nfluence of mmediate and Delayed nsemination on Fertilization of Preovulatory Eggs mmediate Delayed insemination insemination Preovulatory eggs Fertilized 154 (54.0% 256 (71.9% eggs). eggs) 21 (P < 0.001) Not fertilized 131 (46.0% 100 (28.1% eggs) eggs) x' Fertility and Sterility

5 Table 5. The nfluence of mmediate and Delayed nsemination on the Onset of Cleavage Fertilized eggs Embryos transferred Failed cleavage anot significant. mmediate Delayed insemination insemination X' (72.1% fer- 178 (69.6% fertilized) tilized) 43 (27.9% fer- 78 (30.4% fertilized) tilized) n the first 4 months of the year, CC, 100 mg daily, was administered from day 3 of the cycle until the leading follicle was> 14 mm in diameter. At this stage, 300 V of hmg was administered daily until the leading follicle was> 18 mm in diameter. The hmg treatment was then stopped; and after a coasting period of ~ 60 hours, hcg was administered. Over the next 4 months, 100 mg of CC was administered from days 3 to 9 of the menstrual cycle; and hmg, 300 V daily, was commenced on day 7 of the cycle. The hmg was continued until the leading follicle attained a diameter of 18 mm. Again, hcg was given after a 60-hour interval from the last injection of hmg. n the last few months of the year, the above procedure was maintained except that the daily dosage of hmg was reduced to 150 V. During this period, the coasting interval from the last hmg injection to hcg administration was sometimes decreased to 36 hours. As in the previous protocols, plasma E2 levels were used in conjunction with follicular size and numbers so that the day of the menstrual cycle on which patients were tq be admitted to the hospital for LH monitoring and for timing of the hcg injection could be determined. Protocol V n this schedule, devised by Dr. John McBain, CC, 50 to 100 mg daily, was used in combination with hmg, 75 to 300 V daily; in some cases, hmg was administered without CC. The main features of this regimen were that both the length of individual menstrual cycles and the ovarian response to therapy guided the type and duration of treatment. n this individualized approach to ovarian stimulation, the daily E2 levels, ultrasonographic follicular diameter measurements, cervical mucus changes, and the day of the menstrual cycle were integrated. CC was commenced on days 3 to 4 in cycles of 24 to 26 days' duration, and on days 5 to 6 in 28- Vol. 40, No.3, September 1983 to 30-day cycles. Generally, CC was continued for 5 to 7 days, depending on the ovarian response. On the last day ofcc therapy, the size of the most advanced follicles, measured by ultrasound, ranged from 8 to 16 mm in diameter. The commencement and dosage of hmg was based on the ultrasonographic diameter of the secondary follicles, the aim being to prevent atresia in this subgroup of the responding cohort of follicles. For example, if the second-order follicles in the course of CC therapy were 8 to 12 mm in diameter, 225 V of hmg would be given, but if their diameter was 12 to 16 mm, the dose of hmg was 150 V. The hmg was maintained until at least one of these follicles attained a diameter of 18 mm. When the secondary follicles were boosted to develop satisfactorily, the diameter of the leading follicle was usually not more than 4 to 5 mm larger. The average duration ofhmg administration was about 3 days. When this regimen produced the expected increase in follicular diameters and a concomitant progressive increase in plasma E 2, hcg was injected 36 to 48 hours after the last dose of hmg. On the other hand, if follicles continued to en-' large in the face of falling E2 levels, the cycle was abandoned. RESULTS The results obtained with the four different ovarian stimulation protocols are compared in Table 7. The use ofhmg in combination with CC produced a larger average number of follicles per patient, compared with that in the group treated with CC alone. This resulted in a higher yield of preovulatory eggs from the two groups receiving CC and gonadotropin. t is of interest, however, that the fertilization rate of eggs retrieved from patients treated with CC alone was significantly higher than that of eggs from the CChMG group Table 6. The nfluence of mmediate and Delayed nsemination of Preovulatory Eggs on the Blastocyst mplantation Rate mmediate insemination Embryos trans- 111 ferred Blastocysts implanteda embryos) 3 (2.7% Failed implantation 108 (97.3% embryos) Delayed insemination (9.5% embryos) 161 (90.5% embryos) 3.9 (P < 0.05) aelevated ~-hcg in urine; only four term pregnancies were established in 1980/1981. Lopata Concepts in VF -ET 293

6 Table 7. Comparison of Egg Recovery, Fertilization, ET, and the Outcome of Pregnancies in Four Ovarian Stimulation Protocols Used in 1982 Protocol Protocol V Protocol Protocol (CChMG set (CChMG individ (CC days 5 to 9) (extended CC) regimen) ualized) Total Laparoscopies Follicles aspirated 129 (3.0lapa) 461 (4.llap) 950 (6.4/lap) 386 (5.6/lap) 1926 (5.2/lap) Eggs recovered 75 (1.7/lap) 294 (2.6/lap) 734 (5.0/lap) 275 (4.0/lap) 1378 (3.7/lap) Laparoscopy yielding at least 35 (81.4%) 103 (92.0%) 143 (96.6%) 65 (94.2%) 346 (93.0%) one egg Eggs inseminated Eggs fertilized 56 (75.4%) 200 (71.2%) 432 (59.6%) 149 (57.8%) 837 (63.1%) Laparoscopy yielding at least 31 (72.1%) 91 (81.3%) 127 (85.8%) 50 (72.5%) 299 (80.4%) one fertilized egg Embryos transferred Transfer patients 27 (62.7% lap) 84 (75.0% lap) 119 (80.4% lap) 42 (60.9% lap) 272 (73.1% lap) Total pregnancies 4 (9.3% lap) 13 (11.6% lap) 22 (14.9% lap) 16 (23.2% lap) 55 (14.8% lap) (14.8% tr patb) (15.5% tr pat) (18.5% tr pat) (38.1% tr pat) (20.2% tr pat) Ongoing pregnancies 2 (4.7% lap) (7.4% tr pat) 5 (4.5% lap) (6.0% tr pat) 13 (8.8% lap) 10 (14.5% lap) (10.9% tr pat) (23.8% tr pat) 30 (8.1% lap) (11.0% tr pat) alaparoscopies. btransfer patients. (71.9%, compared with 59.1%; X:.! = 17.4, P < 0.001). t is also noteworthy that a significantly higher proportion of fertilized eggs underwent normal cleavage in the total CC group, compared with the total CC/hMG group (84.8% versus 75.4%; X2 = 8.64, P < 0.005). There was no significant difference in fertilization rate or cleavage rate between the two CC groups or between the two CChMG groups. Despite the significantly lower fertilization and cleavage success rates in the CC/hMG group, there was no significant difference between the percentage of patients receiving ET in the CC alone (71.6%) and the gonadotropin-supplemented (74.2%) groups, nor was there a significant difference in the overall pregnancy success rate between these two groups. Moreover, although the percentage of continuing and term pregnancies in the total CChMG group was more than double that in the CC alone group (14.3% versus 6.3%; X2 = 3.5,0.05 < P < 0.10), the difference in proportions was insignificant, because of the low numbers in each group. Of the two CChMG protocols used, the individualized treatment regimen (protocol V) has produced a significantly higher proportion of pregnancies per transfer patients (38.1 % versus 18.5%; X2 = 5.6, P < 0.02). However, there was no significant difference between the percentages of ongoing pregnancies in these two groups. The results have been grouped and analyzed in terms of the ovarian stimulation protocols employed. n view of the fact that different stimula- 294 Lopata Concepts in VF -ET tion regimens may produce similar ovarian responses, as judged by ovarian follicular development and plasma E2 levels, it was thought that it would be informative to analyze the data entirely on the basis of the type of ovarian response elicited. After all, we should aim at inducing and recognizing the optimal ovarian response irrespective of whether CC alone, CChMG, or hmg alone is used for producing fertile eggs. n evaluating the results with respect to the ovarian response, two major parameters were used to group patients. One of these was the level of plasma E2 per preovulatory follicle (15 mm or more in diameter) elicited by the treatment, and the other was the timing of the hcg injection in relation to the E2 peak achieved in a particular patient. Thus, one group comprised those patients in whom the hcg was injected before the observed plasma E2 peak, and the second group comprised those in whom the hcg was injected at or after the peak E2 level. Each of these two groups was further subdivided into the "high" and "low" responders, i.e., those who achieved a plasma E2 level per preovulatory follicle of;;;.: 500 pg/ml and < 500 pg/ml, respectively. n addition, those in whom an endogenous LH surge was observed were analyzed separately but again subdivided into high and low responders. t is evident that this sort of classification generates at least six different groups for statistical analysis. For each of these groups a comparison of such important parameters as the oocyte recovery rate, fertilization rate, cleavage success rate, ET Fertility and Sterility

7 Table 8. The Fertilization of Preovulatory Eggs in Relation to the Timing of hcg Administration Relative to the Plasma E2 Peak and the Magnitude of the Peak hcg given hefore plasma E. P'!ak (pglmllfollicle) hcg given at or after plasma E. P'!ak (pg/mllfollicje) Spontaneous LH surge (E. = pg/mj/follicje) Combined groups (E. = pg/mllfollicle) E. < 500 E.;. 500 Whole E. < 500 E.;. 500 Whole E. < 500 E.;. 500 Whole Total Total group group group E. < 500 E.;. 500 Group" A, A2 Ag B, B. Bg C, C2 Cg A,+B,+C, A2+B2+C2 Eggs insem inated Eggs fer tijized FertiJiza tion rate (%) achi-square analysis of groups (NS, not significant): A, versus A2, P < 0.005; A, versus B " NS; A2 versus B2, P < 0.001; A3 versus B3, P < 0.001; B, versus B2, NS; A, versus Cl> NS; A2 versus C2, NS; A3 versus C3, NS; C, versus C2, NS; B, versus C " NS; B2 versus C2, NS; B3 versus C3, P < 0.005; (A, + B, + C,) versus (A2 + B2 + C2), P < success rate, and total and ongoing pregnancy success rates can be evaluated. Because too many tables would thus be generated for this review, only two are included (Tables 8 and 9), although the more important findings derived from an analysis of the other tables will be described. Oocyte Recovery There was no significant difference in the oocyte recovery rate per follicle between the patients receiving hcg prior to the plasma E2 peak and those given the gonadotropin at or after the peak. However, both of these groups produced a significantly higher yield of oocytes per follicle, compared with the group having an endogenous LH surge; in this group, the patients with the high plasma E2 values (~ 500 pg/mllfollicle) were found to have the lowest recovery rate. n contrast, when the final stages of follicular maturation were controlled by hcg, the patients with high E2 levels produced the best oocyte recovery rates. Fertilization Rate (Table 8) t should be remembered that, in our program, oocytes were aspirated 36 hours after the hcg injection and that all preovulatory eggs were incubated for 3 to 6 hours before sperm were added. The results summarized in Table 8 show that under these conditions the fertilization rate was significantly higher if oocytes were recovered from patients given hcg - 12 to 24 hours before the plasma E2 peak (Fig. 3). n patients with a spontaneous LH surge, oocytes were aspirated at 24 to 26 hours after the increase in urinary LH. n this group, the fertilization rate (70.2%) was significantly higher than in the group receiving hcg at or after (Fig. 4) the E2 peak (52.6%). However, there was no significant difference between the fertilization rates in the group with an endogenous LH surge (Fig. 5) and the group given hcg prior to the E2 peak. Another point of interest is that the combined high E2 groups (A2 + B2 + C2 in Table 8) produced eggs with a significantly higher fertilization rate than the combined low E2 groups (A + Bl + C 1). Table 9. The Pregnancy Success Rate in Relation to the Timing of hcg Administration Relative to the Plasma E2 Peak and the Magnitude of the Peak hcg given hefore plasma E2 peak (pg/mllfollicle) hcg given at or after plasma E2 P'!ak (pg/mllfollicje) Spontaneous LH surge (E. = pg/mllfollicle) Combined groups (E2 = pg/mllfolliclej E. < 500 E2 ;. 500 Whole E2 < 500 E2 ;. 500 Whole E2 < 500 E2;' 500 Whole Total Total group group group E2 < 500 E. ;. 500 Group" A, A2 Ag B, B2 Bg C, C. Cg A,+B,+C, A2+B.+C. Laparoscopies Pregnancies Pregnancy rate (%) achi-square analysis of groups (NS, not significant): A, versus A2, NS; A, versus Bl> P < 0.02; A2 versus B2, NS; A3 versus B3, P < 0.02; B, versus B2, NS; A, versus C " NS; A2 versus C2, NS; A3 versus C3, NS; C, versus C2, NS; B, versus Cl> P < 0.05; B2 versus C2, NS; B3 versus C3, NS; (A, + B, +C, ) versus (A2 + B2 + C2), NS. Vol. 40, No.3, September 1983 Lopata Concepts in VF -ET 295

8 800 E ~ E2 Peak.5OO pg/ml/foll - - E2 Peak (500 pg/ml/toll... ~ Total Means t SO (from logarithmic data) HCG NJECTED BEFORE E, PEAK Days Figure 3 The mean daily plasma E2 levels per follicle relative to the day of hcg injection. The top graph shows the arithmetic means of plasma E2 for 59 high responders (E2 peak > 500 pg/mllfollicle > 14 mm in diameter). The bottom graph shows the arithmetic means of plasma E2 for 85 low responders (E2 peak < 500 pg/mllfollicle > 14 mm in diameter). The middle graph represents the overall plasma E2 means for high and low responders (combined total, 144) ± 1 standard deviation computed from logarithmic data. LAP, laparoscopy for oocyte collection; ET, embryo transfer. 5 Pregnancy Success Rates (Table 9) The pregnancy success rates in different groups, in terms of laparoscopies carried out, are shown in Table 9. With the current numbers, the clearest outcome was that there was a significantly higher pregnancy rate in patients given heg on the day before the plasma E2 peak, compared with patients in whom the heg was delayed until E2 peaked or started to drop. n this group, the patients with low plasma E2 levels «500 pg/mll follicle) had the lowest pregnancy rate. However, when the combined low E2 group was compared with the combined high E2 group, there was no significant difference in pregnancy rates. The same trends as described above were evident when pregnancy rates were calculated in terms of transfer patients. n the group given heg before the E2 peak, in the group with delayed heg, and in the spontaneous LH surge group, the total pregnancy rates were 22.9%, 9.8%, and 20.0%, respectively. n 1982 a total of 55 pregnancies were obtained following VF-ET (some of these are not included in Table 9 because of incomplete plasma E2 data). Of the total pregnancies established, 30 are ongoing or term pregnancies, whereas the others did Cleavage Success Rate The cleavage success rate in eggs derived from the group of patients having an endogenous LH surge was significantly higher than the cleavage success rate of fertilized eggs in the other two groups. A comparison of the latter groups indicated that cleavage was significantly higher in eggs retrieved from patients given heg prior to the plasma E2 peak, compared with eggs derived from patients given the heg at or after the E2 peak. There was no significant difference in cleavage success rates when the high and low plasma E2 groups were compared. Patients Receiving Embryo Transfer The percentage of patients receiving ET, expressed in terms of laparoscopies carried out, was significantly higher in the group given heg while the E2 level was rising, compared with those given heg at or after the E2 peak (79.4% versus 64.6%). However, there was no significant difference between the spontaneous LH surge group (68.2%) and the other two groups, nor were there any differences between the high and low plasma E2 groups. 296 Lopata Concepts in VF -ET 800 HCG NJECTED AT OR AFTER E, PEAK [,!!800.1/ ~ r i 1400~ ~ - E, Peak'500pg/mlifoll -- E2 Peak (500 pg/mltfoll... E2 Total Means t SO (from logarithmic datal,,, Days, -2,,,,, t HCG LAP ET Figure 4 The mean daily plasma E2 levels per follicle relative to the day of hcg injection. The top graph shows the arithmetic means of plasma E2 for 21 high responders (E 2 peak > 500 pg/mllfollicle > 14 mm in diameter), The bottom graph shows the arithmetic means of plasma E2 for 44 low responders (E2 peak < 500 pg/mllfollicle > 14 mm in diameter), The middle graph represents the overall plasma E2 means for high and low responders (combined total, 65) ± 1 standard deviation computed from logarithmic data. LAP, laparoscopy for oocyte collection; ET, embryo transfer. Fertility and Sterility

9 BOO 800, -7 - E, Peak> 500 pg/ml/toll -_. E, Peak <500 pg/ml/foll... E2 Total Means±SD (from logarithmic data) SPONTANEOUS LH SURGE,,,, Days, -1 0 LH Surge Figure 5 The mean daily plasma E2 levels per follicle relative to.the day of the urinary LH surge. The top graph shows the arthmetic means of plasma E2 for 19 high responders (E2 peak> 500 pg/mllfollicle > 14 mm in diameter). The bottom graph shows the arithmetic means of plasma E2 for 19 low responders (E2 peak < 500 pg/mllfollicle > 14 mm in diameter). The middle graph represents the overall plasma E2 means for high and low responders (combined total, 38) ± 1 standard deviation computed from logarithmic data. LAP, laparoscopy for oocyte collection; ET, embryo transfer. not survive for various reasons (Table 10). f the progressing and term pregnancies were distributed between the different groups being evaluated, the numbers at present would be too small for adequate statistical analysis. LAP MPLCATONS OF CURRENT RESULTS The major advantages of using hmg in combination with CC, in preference to CC alone, include the following: (1) More secondary follicles are boosted to the preovulatory stage, and hence more eggs become available per patient. (2) More embryos became available for transfer per patient. Some of the implications of these findings will be briefly evaluated. MULTPLE EGGS Table 11 compares the fertilization and cleavage success rates in relation to the number of eggs obtained per patient. Patients in whom nine or more eggs were recovered (preovulatory in appearance as judged by the presence of a sticky cumulus) had a significantly lower fertilization and cleavage success rate than those yielding one to four preovulatory eggs. Similarly, the group ET 5 yielding five to eight eggs had a significantly lower fertilization rate than patients in whom one to four eggs were retrieved. The chi-square value for trend showed that there was a significant decrease in the fertilization rate in the three groups (Table 11). Although the cleavage success rate in patients yielding more than nine eggs was significantly lower than in the one- to four- and five- to eightegg groups, the chi-square value for trend was insignificant for the three groups. Table 12 shows the ET and pregnancy rates in relation to the number of eggs obtained per patient. t is of interest that there is no significant difference in the percentage of patients receiving ET, or in the percentage becoming pregnant, in the three groups. The results therefore suggest that when human ovaries are stimulated to produce large numbers of eggs, which are aspirated at about the same time, their overall fertilization and cleavage potential is reduced; yet some of the eggs have the potential for inducing pregnancies. These fertile eggs are difficult to identify, particularly when several cleaving embryos of unknown potential are produced in each patient. n several patients yielding nine or more eggs, some eggs were selected for light- and electronmicroscopic examination. These were selected at random and fixed immediately after their aspiration from follicles. The aims of this study were to assess (1) the maturity of the eggs, (2) the cortical granule organization at the surface of the oocytes, and (3) the state of their plasma membrane and zona pellucida. Of the ten cumulus-enclosed eggs examined, three were at the germinal vesicle stage, one was at meiotic metaphase, and the remaining six had completed the first meiotic division and were at metaphase. Moreover, two of the mature eggs were releasing cortical granule material into the perivitelline space. The plasma membrane of some of these eggs contained breakages, and the inner surface of the zona pellucida appeared to be undergoing dissolution. These results suggest that in patients yielding nine or more eggs following treatment with CC hmghcg, up to 60% of the eggs in cumulus may Table 10. The Outcome of 1982 Pregnancies Total pregnancies Term/ongoing Ectopic Clinical abortions Biochemical pregnancies y Vol. 40, No.3, September 1983 Lopata Concepts in VF -ET 297

10 Table 11. Fertilization and Cleavage" in Relation to the Number of Eggs Obtained per Patient No. of Eggs eggs/patient inseminated ~,s fe' lzed Eggs cleaving (69.1%) 350 (80.6%) (60.8%) 206 (79.8%) ~9 : (56.1%) 114 (70.8%) afertilization: X2 for trend = 5.895, P < Cleavage: X2 for trend = 1.167, P = be either immature or undergoing degenerative changes. This may result in abnormal or failed fertilization in, a high proportion of the eggs. t should be noted that at the present time we do not have the means for deciding how long eggs should be incubated before they are inseminated. The routine 3 to 6 hours of preincubation may be inadequate for meiotically immature eggs, and such an interval may be unsuitable for oocytes in which spontaneous cortical granuleexocytosis is occurring. MULTPLE EMBRYOS An improved chance of establishing pregnancy is the benefit of multiple ET; an increased incidence of multiple pregnancy, and possibly of ectopic pregnancy, is the risk. The fate of any spare embryo is the moral dilemma. Table 13 shows the relationship between the number of embryos inserted into the uterus and the resulting pregnancy rates. The pregnancy success rate, in terms of patients undergoing ET, was just above 11 % when single embryos were transplanted; and it progressively increased to> 38% when four embryos were inserted into the uterus. The rise in the pregnancy rate with increasing numbers of embryos transferred was found to be highly significant (X 2 for trend = 9.0, P < 0.003). t should also be noted that nine twin pregnancies were established (Table 13). This represents a twinning rate of 30% in the group of patients with ongoing pregnancies. t would appear, therefore, that multiple ET increased the twinning rate by more than 30 times, compared with the dizygotic twinning rate in the general population (frequency, 0.7% to 1.0%). However, the multiple pregnancy rate following the transfer of two to five embryos is remarkably similar to that reported for anovulatory patients treated with gonadotropins (frequency, 20% to 30% in recent series. Thirteen series have been reviewed by Brown 13 ). n a recent paper 14 dealing with the risks and benefits of multiple ET, the probability ofproducing triplets and quadruplets has been evaluated. t has been predicted that the probability of triplets is 0.4% of ongoing pregnancies following the transfer of three embryos and 1% when four embryos are inserted. The probability of quadruplets following the placement of four embryos in the uterus was calculated to be 0.03% of continuing pregnancies. f these predictions are correct, the incidence of triplets following the transplantation of three or four embryos would be 40 to 100 times greater than that occurring in the general population (frequency, 0.01%). t is currently unknown whether the transfer of multiple embryos predisposes to ectopic pregnancy. n 1982 in our unit, ectopic pregnancy occurred in six patients (Table 10). Of these, two received four embryos, two received three, one received two, and one patient received a single embryo. THE SPARE EMBRYO Table 12. ET and Pregnancy Rate in Relation to the Number of Eggs Obtained per Patient No. of eggs/patient ~9 Laparoscopies achi-square for trend, not significant. Transfer patients 183 (75.3%) 63 (82.9%) 23 (85.2%) Nsa n establishing our policy for aspirating all available preovulatory oocytes from each patient, two major issues were taken into consideration. First, it was recognized that it is impossible to assess, at the time of laparoscopy, which follicles contain eggs capable of producing pregnancy. Second, it was known that the placement of up to four embryos in the uterus was associated with a high pregnancy rate (Table 13); and although the incidence of twinning was about 35%, there was an acceptably low likelihood oftriplets (1% ofvf pregnancies) or quadruplets (0.03% of VF pregnancies). However, with the transfer of five or more em. bryos, we approach a state where the risks of a Pregnancies % Laparoscopies % Transfer patients NS NS 298 Lopata Concepts in VF -ET Fertility and Sterility

11 7 ) f Table 13. The nfluence of Multiple ET on Pregnancy Rate and Type of Pregnancy (Laparoscopies 1-372, 1982) No. of Type of pregnancy embryos Transfer Pregnancies transferred patients Single Twin (11.2%) (18.6%) 12 2 a (22.6%) 13 l a (38.6%) (14.3%) 1 0 aone fetus was resorbed after two fetal hearts were detected in one of the patients. X 2 for trend (1, 2 or 3, 4 or 5 embryos transferred) = 9.0, P < high incidence of multiple pregnancy probably outweigh the benefits of multiple ET. For example, even if we assume that only 10% of transferred embryos produce pregnancies in a receptive uterus, it can be shown that the expected incidence of triplets following the placement of five embryos would be 200 times that found in the general population. This frequency rises markedly with increasing numbers of embryos introduced into the uterus. Since five or more preovulatory eggs are obtained in about 30% of laparoscopies, spare embryos will arise in some cases if the preferential policy is to transfer a maximum of four morphologically normal embryos. The moral and legal status of the preimplantation human embryo is being discussed extensively,15-17 but clear principles and guidelines regard -ing the fate of the extrauterine embryo are yet to be established. The main difficulty arises from the claim being made by some religious and minority groups that the preimplantation human embryo deserves the same rights and protection as a human subject on the grounds that the embryo is a potential human being. The arguments for and against these views have been reviewed by others.15 Because every embryo that is not placed in the uterus is destined to perish (90% of those inserted into the uterus fail to give rise to term pregnancies), and because biologic development is a continuum of increasing complexity, the spare embryo must be regarded at the present time as human tissue of limited viability. As such, decisions must be made as to who owns the spare embryo, who is responsible for its fate, and whether carrying out ethically regulated scientific studies upon the embryonic tissue is morally more desirable than the act of throwing it away. At present, our edict is to allow the embryo to succumb and then to discard it. This philosophic stance may be safe but leads to the loss of infor- mati on that could benefit patients and the whole field of VF-ET. COMMENTARY AND CONCLUSONS The significantly higher fertilization rate and embryo yield in 1981, compared with 1980, is likely to be due to two main factors. These include the larger average follicular diameter used for timing the heg injection and the delayed insemination of preovulatory oocytes, both of which were introduced in the second half of When the effects of each of these factors were examined separately, it was found that delayed insemination did not significantly increase the fertilization rate of eggs derived from leading (largest) follicles. However, it was found that when preovulatory eggs derived from secondary follicles were preincubated for up to 6 hours before insemination, their fertilization rate was significantly greater than that of equivalent eggs inseminated immediately after recovery. t would appear, therefore, that the higher fertilization rate observed in 1981 was largely due to the delayed insemination, which significantly enhanced the fertilizability of eggs derived from second-order follicles. This effect may be due to progressive oocyte maturation during the incubation interval. 1S t is also of interest to note that the increased yield of embryos following delayed insemination was due to the increased percentage of eggs undergoing fertilization rather than an increased cleavage success rate of fertilized eggs. That is, the 3- to 6-hour preincubation interval does not appear to influence the initiation of cleavage of pronucleate eggs (Table 6). n contrast to this, our findings suggest that embryos derived from pre incubated.eggs have a significantly higher chance of producing blastocysts capable of implantation (Table 6). Studies to determine whether preincubation of oocytes leads to a higher incidence of ongoing pregnancies have not been done. The work carried out in 1980 and 1981 has indicated that even with the most accurate ultrasound monitoring of follicular growth and size, the measurement of leading diameters can only provide an approximate indication of follicular maturity and quality. For example, eggs derived from follicles ranging from 15 mm to 35 mm in diameter have given rise to viable embryos and term pregnancies. y Vol. 40, No.3, September 1983 Lopata Concepts in VF -ET 299

12 Although the CC/hMG therapy produced a larger number of follicles and eggs per patient, their overall fertilization and cleavage success rates were significantly lower than that of eggs derived from patients treated with CC alone. There was also a significant decrease in the fertilization rate with increasing numbers of eggs obtained per patient (Table 11). These findings are likely to be due to the asynchrony of follicular development19 or anomalies of follicular growth and maturation2o induced by hyperstimulation. t would appear, for example, that there was a breakdown of synchrony between follicular and meiotic maturation, indicated by the aspiration of cumulus-enclosed eggs that were at the germinal vesicle stage, in patients hyperstimulated to yield nine or more oocytes. The finding that there was no significant difference in the pregnancy rate between patients yielding one to four, five to eight, or nine or more eggs indicates that there are likely to be viable, immature, and atretic oocytes in a cohort of any size. The challenge for the future is to devise a procedure that can identify follicles of different quality within the cohort. An even more useful breakthrough, however, would be to develop an ovarian stimulation program that optimizes the synchrony between follicles in the developing cohort. Of the two CC/hMG protocols, only the individualized treatment regimen (protocol V) produced a significantly higher percentage of total pregnancies and continuing pregnancies than the combined CC alone group. Although the treatment based on the individualized ovarian response produced a higher percentage of pregnancies than the predetermined treatment (protocol ), there was no significant difference in the percentage of continuing pregnancies in these two groups. At the present time, therefore, the low numbers in each group make it premature to draw conclusions between the two different CC hmg protocols. t is also worth noting that the abortion rate in the two CClhMG groups was - 40%, whereas that in the combined CC alone group was - 60%. When the results were assessed on the basis of the timing of hcg administration and the E2 response to ovarian stimulation, it was found that the best results were obtained in the group of patients given hcg shortly before the E2 peak and in the group in which oocyte collection was timed from the onset of a spontaneous LH surge. Thus, the fertilization rate, the cleavage success rate, the percentage of patients receiving ET, and the total pregnancy rate were higher in the group of patients given hcg on the day before the estrogen peak, compared with the group in which the gonadotropin injection was delayed until or after the peak level. Similarly, the group with a spontaneous LH surge performed better than the delayed hcg group, although the numbers were generally too low for adequate statistical evaluation. The reason for the poorer performance in the group in whom the hcg is postponed is unknown. t is possible that in some cases, in this group, the release of endogenous LH has been suppressed,21,22 and the hcg injection is given too late for normal synchronization of follicular and oocyte maturation. The magnitude of the E2 response appears to influence the fertilization rate but none of the other parameters. t was found, for example, that there was a significantly higher fertilization rate in the combined high E2 group, compared with the low E2 group; yet there were no significant differences in the cleavage success rate, the percentage of patients receiving ET, or the pregnancy rate, between the high and low responders. The enhancement of the fertilization rate by high follicular E2 levels has been described previously.23 By far the clearest outcome of the 1982 study was the highly significant increase in the pregnancy success rate with the increasing number of embryos transferred into the uterus. Based on the data reported at recent VF meetings, it would appear that these results will be confirmed by the Monash University team and the Eastern Virginia Medical School team at Norfolk. However, the exact incidence and types of multiple pregnancy following the placement of up to four embryos in the uterus need to be evaluated over the next few years. n 1982 the embryos produced in our VF program appeared to have a 10% to 12% chance of producing term pregnancies. On the basis of this estimate, the benefits of transferring up to four embryos may be considered to outweigh the risks of multiple pregnancy. As if to highlight the risks, however, two triplet pregnancies were diagnosed as this paper was being written. n one patient, three intrauterine fetal hearts have been detected following the insertion of three embryos, and in the second patient, three intrauterine gestational sacs have been detected following the placement of four embryos. The occurrence of 300 Lopata Concepts in VF-ET Fertility and Sterility

13 these two triplet pregnancies early this year means that we will need to reexamine our assumptions about the low survival potential of the embryos we are currently producing. f the endocrine and laboratory procedures introduced in 1983 have dramatically improved embryo quality, we will need to reassess all the principles underlying multiple ET. Acknowledgments. The work described in this paper could not have been carried out without the collaboration and valuable contribution of the following members of our VF team: Dr. an Johnston, the director of the Reproductive Biology Unit and chief clinician in the VF program; Professor Roger Pepperell and Professor James Brown, who advised us on the endocrine treatment of patients; Dr. Marion Martin, who supervised the endocrine monitoring; our three laparoscopists, Dr. John McBain, who also devised a highly successful ovarian stimulation program, Dr. Michael Gronow, who coordinated the entire program, and Dr. Andrew Speirs, who also provided us with data analysis facilities. Dr. Geoffrey Kellow, Dr. Peter Leung, and Miss Yvonne du Plessis assisted me in the laboratory. Dr. Hugh Robinson and Dr. Lach de Crespigny performed the ultrasound examinations. Mr. David Hay and Dr. Pat Tasker carried out the rapid LH and ~-hcg assays. Mrs. Anne McCartin maintained the communication links with patients and provided excellent secretarial assistance. REFERENCES 1. Beier H, Lindner H (Eds): Fertilization of the Human Egg in Vitro: Biological Basis and Clinical Applications. Heidelberg, Springer-Verlag, Crosignani PG (Ed): n vitro fertilization and embryo transfer. n Serono Clinical Colloquia on Reproduction, No.4. New York, Academic Press, n press 3. Speirs AL, Trounson A, Warnes GM, Yovich J, Saunders 0, Chen C: Summary of results. n Clinical in Vitro Fertilization, Edited by C Wood, A Trounson. Berlin, Springer-Verlag, n press 4. Edwards RG: n Proceedings of the First Bourn Hall Meeting on Human Conception in Vitro, Edited by RG Edwards, JM Purdy. London, Academic Press, Lopata A, Wood C: n vitro fertilization and embryo transfer: its clinical application. n Oxford Reviews of Reproductive Biology, Vol 7, Edited by CA Finn. Oxford, Oxford University Press, 1982, p Lopata A, Johnston, Speirs A: n vitro fertilization. n Current Therapy of nfertility, Edited by CR Garcia, L Mastroianni, RD Amelar, L Dubin. Trenton, NJ, B. C. Decker, 1982, p Lopata A, McBain JC, Johnston WH, Speirs AL: n vitro fertilization and embryo implantation in the treatment of infertility. n Progress in Gynecology, Vol 7, Edited by ML Taymor. New York, Grune & Stratton, n press 8. Lopata A, Johnston WH, Hoult J, Speirs AL: Pregnancy following intrauterine implantation of an embryo obtained by in vitro fertilization of a preovulatory egg. Fertil Steri33:117, Leeton J, Trounson A, Jessup 0, Wood C: The technique for human embryo transfer. Fertil Steril 38:156, Craft, Bernard A, Djahanbakhch 0, McLeod F: Embryo transfer catheter material. Lancet 1:680, Kerin JFP, Warnes GM, Jeffrey R, Cox LW, Broom TJ: A simple technique for human embryo transfer into the uterus. Lancet 2:726; Johnston WH, Speirs AL, Gronow MJ, McBainJC, Lopata A: Clinical aspects of in vitro fertilization and embryo transfer. n Serono Clinical Colloquia on Reproduction, No.4, Edited by PG Crosignani. New York, Academic Press, n press 13. Brown JB: Gonadotrophins. n nfertility: Male and Female, Edited by V nsler, B Lunenfeld. London, Churchill Livingston, n press 14. Speirs AL, Lopata A, Gronow MJ, Kellow GN, Johnston WH: Analysis of the benefits and risks of multiple embryo transfer. Fertil Steril 39:468, Walters L: Human in vitro fertilization: a review of the ethical literature. Hastings Cent Rep 9:23, Studdard PA: Current issues in in vitro fertilization. Ala J Med Sci 18:184, Grobstein C: The moral uses of "spare" embryos. Hastings Cent Rep 12:5, Trounson AO, Mohr LR, Wood C, Leeton JF: Effect of delayed insemination on in vitro fertilization, culture and transfer of human embryos. J Reprod Fertil 64:285, Hodgen GO: Oocyte transfer and fertilization "in vitro." n Serono Clinical Colloquia on Reproduction, No.4, Edited by PG Crosignani. New York, Academic Press, n press 20. Hunter RHF, Cook B, Baker TG: Dissociation of response to injected gonadotropin between the Graafian follicle and oocyte in pigs. Nature 260:156, Brown JB: Unpublished data 22. Jones HW Jr, Jones GS, Andrews MC, Acosta A, Bundren C, Garcia J, Sandow B, Veeck L, Wilkes C, Witmyer J, Wortham JE, Wright G: The program for in vitro fertilization at Norfolk. Fertil Steril 38:14, Carson RS, Trounson AO, Findlay JK: Successful fertilization of human oocytes in vitro: concentration of estradiol-17~, progesterone and androstenedione in antral fluid of donor follicles. J Clin Endocrinol Metab 55:798, 1982 Received April 11, Reprint requests: Alexander Lopata, M.B., B.S., Ph.D., Department of Obstetrics and Gynaecology, University of Melbourne and Reproductive Biology Unit, Royal Women's Hospital, Melbourne, Victoria, Australia. Vol. 40, No.3, September 1983 Lopata Concepts in VF -ET 301