Evo l ution of a h i gh l y successfu l i n vitro ferti l ization-embryo transfe r program

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1 Vol. 48, No. 1, July 1987 FERTILITY AND STERILITY Copyright e 1987 The American Fertility Society Printed in U.S.A. Evo l ution of a h i gh l y successfu l i n vitro ferti l ization-embryo transfe r program David R. Meldrum, M.D.* Ryszard Chetkowski, M.D. t Kenneth A. Steingold, M.D.* Dominique de Ziegler, M.D. Marcelle I. Cedars, M.D. Minda Hamilton, B.S. Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology, University of California, Los Angeles (UCLA) School of Medicine, Los Angeles, California During the first 2! years of operation of the University of California at Los Angeles in vitro fertilization-embryo transfer program, 47 clinical pregnancies were achieved in 154 laparoscopies for oocyte aspiration ( 3 1 % ). Two of these pregnancies were achieved through transfer of cryopreserved embryos when ongoing pregnancy did not result from embryo transfer in the stimulated cycle. An increase in clinical implantation was noted with a reduction of transfer volume and proportion of air, with 34% of laparoscopies being followed by clinical pregnancy over the last 18 months. No difference in the rate of clinical pregnancy was noted relative to uterine depth or position. The high rate of multiple implantation (47%) suggested a high level of embryo quality. The success rate was attri buted to a strong ovarian, human menopausal gonadotropin stimulation, accurate timing of human chorionic gonadotropin, a high oocyte retrieval rate, meticulous laboratory technique, atraumatic, high-fundal transfer of embryos, and initiation of embryo cryo preservation. Fertil Steril 48:86, 1987 Since there is no universally accepted set of tech niques for in vitro fertilization-embryo transfer (IVF-ET), a new program is usually established by patterning all procedures and instrumentation after one successful facility. Our approach prior to beginning the program at the University of Califor nia at Los Angeles (UCLA) in September 1983 was to examine the techniques used by several teams that had established a high rate of success in an Received November 18, 1986; revised and accepted March 1 1, * Reprint requests: David R. Meldrum, M.D., In Vitro Fertil ization Center, AMI South Bay Hospital, 514 North Prospect Avenue, Redondo Beach, California t Present address: Department of Obstetrics and Gynecology, Alta Bates Hospital, Berkeley, California. :j: Present address: Department of Obstetrics and Gynecology, Medical College of Virginia, Richmond, Virginia. Present address: Department of Obstetrics and Gynecology, University of Medicine and Dentistry New Jersey Medical Center, Newark, New Jersey. 86 Meldrum et al. Evolution of an IVF-ET program attempt to draw on what appeared to be the strengths of each program. It is the purpose of this report to describe the techniques we have used and the results achieved over the 2! years of operation. MATERIALS AND METHODS Between September 20, 1983 and March 20, 1986, 154 attempts were made at oocyte retrieval by laparoscopy and 14 attempts (8.3% of total cycles) by ultrasound-directed aspiration. Choice of the latter technique was generally based on poor laparoscopic accessibility of the ovaries. A detailed analysis of those cycles will be the subject of an other report after further experience with the tech nique. In most cases, screening laparoscopy was not performed because of the recognition that, in almost all patients, an adequate attempt at oocyte aspiration could be achieved and because the ul trasound technique was available as a back-up. All laparoscopies, ultrasounds, and embryo transfers Fertility and Sterility

2 were done by a single faculty person in conjunction with sequential fellows. The indications for IVF ET, in varying combinations, were tubal (76%), endometriosis (23%), and idiopathic (4%). Twenty-three cycles (15%) had an associated male factor (<20 X 106/ml, <40% motility, <50% normal forms or a subnormal hamster egg penetration test) and 18 attempts (12%) were in women with infrequent (interval > 33 days) menstrual cycles. Five women had uterine factors (three myomata 8 weeks or greater, one diethylstilbestrol-exposed, one prior lysis of intrauterine adhesions). Median age of the female partner was 34 years (range, 24 to 40 years). Women over 40 at the time of evaluation were not accepted. Prior to initiating a treatment cycle, semen was evaluated for pyospermia and appropriate treatment was given. Routine semen culture was not done. Ovarian stimulation was initiated by injection of 225 IU of human menopausal gonadotropins (hmg, Pergonal, Serono Laboratories Inc., Randolph, MA) daily starting on day 2 or 3 of the menstrual cycle, 1 provided there was no cystic follicle over 1 em on baseline ultrasound. Because of the unpredictable response to this regimen in women with infrequent menstrual cycles, patients with menses > 33 days apart were given clomiphene citrate (CC) 100 mg daily from day 3 to 7 together with 75 IU of gonadotropins. Detailed analysis of this group of patients will be the subject of another report. 2 On the fifth day of medication, an ultrasound scan was performed and 225 to 375 IU of gonadotropins were then given daily until injection of human chorionic gonadotropin (hcg). The higher doses were chosen if follicle sizes were <1 em or if marked asynchrony among follicles was evident. In the CC cycles, the dose was usually 225 IU, except in one patient with a particularly prominent response in whom the dose was kept at 75 IU. Doses exceeding 225 IU were empirically given in divided doses at 9 A.M. (150 IU) and 7 to 9 P.M. (150 to 225 IU) to even out any luteinizing hormone (LH) effect. The interval between the last gonadotropin injection and hcg was kept to no more than 28 hours and generally to 14 hours.3 Patients responding to stimulation with only a single mature follicle or with a peak estradiol (E2) level < 500 pg/ml were cancelled and the subsequent cycle was started with the simultaneous CC-hMG regimen described previously. The timing of hcg was determined by both daily E2 levels at 9 A.M. (Immune-Estradiol P 2 5, Pantex, Santa Monica, CA) and daily pelvic ultrasound (Philips 2000 sector scanner, Philips Ultrasound Systems, Santa Ana, CA), using the transvaginal approach when needed. When two follicles reached a longest diameter of 1.5 to 1.6 em (1. 7 to 1.8 em for CC-hMG cycles), 10,000 units of hcg was given intramuscularly (IM) that evening. Laparoscopy was performed 34! to 35 hours later (generally at 8 to 11 A.M.). In some cases, if the level ofe2 was less than expected (each follicle 1.4 em or greater = 150 pg/ml, 1.2 or 1.3 em = 100 pg/ml, 1.0 or 1.1 em = 50 pg/ml for hmg-only cycles), hmg was continued for another day. In some cases where the E2 concentration clearly exceeded or lagged behind the expected value as previously calculated, the hcg was given 1 day early or late, respectively. The previous day's E2 sample was always run simultaneously and, if any drop was observed prior to hcg, the cycle was bypassed. 4 No cycles were cancelled based on an E2 drop after hcg. Thirty to 60 minutes before laparoscopy, pelvic ultrasound was repeated. The cycle was cancelled if ovulation was detected, unless only the largest follicle had collapsed, multiple others remained intact, the tubes were distally occluded, and there were not marked pelvic adhesions noted at prior surgery. Laparoscopy was performed under general balanced anesthesia, which was initiated after preparing the patient. Pneumoperitoneum was established with 5% C02 in air after verifying peritoneal placement by direct visualization using a large Verres needle and miniendoscope.5 Aspiration was carried out with a 12-gauge needle lined by a 14- gauge Teflon catheter through a needle trocar placed 6 em above the symphysis in the midline. Oocytes were collected into prewarmed 15-ml Falcon tubes (#2057, Falcon Plastics, Oxnard, CA) through a Silicone bung at 100 mm Hg negative pressure. Follicles were flushed 2 to 5 times, depending on the number of follicles, with equilibrated Ham's F-10 medium (Flow Laboratories, McClean, VA) containing 40 U/ml of sodium heparin. All cul-de-sac fluid was aspirated at the end of the procedure. The follicle aspirates and cul-de-sac fluid were placed immediately into a C02 controlled, humidified, pediatric isolette at 37 C.5 Areas of endometriosis that could be exposed by retraction were fulgurated with bipolar cautery. Back-up ultrasound aspiration was used only once in this series. Oocytes were identified grossly by pouring the follicular fluid into Petri dishes (Falcon #1029) or Vol. 48, No. 1, July 1987 Meldrum et al. Evolution of an IVF-ET program 87

3 by microscopic screening if none was apparent macroscopically. Oocytes were classified as mature, intermediate, or immature.1 After evaluation and washing, the oocytes (one or two) were placed in 1 ml of medium in prerinsed 5-ml culture tubes (Falcon #2003). Except for immature oocytes, 7 to 8 hours of preincubation were allowed before adding sperm. Immature oocytes were incubated for 24 to 32 hours, depending on the visualization of a polar body.6 Sperm were washed twice 30 minutes after ejaculation with Ham's F-10 medium containing 10% maternal serum and centrifugation at 250 X g. A subpopulation of sperm that were 95% to 100% motile was harvested by drawing off the upper milliliter of 2.5 ml of medium that had been layered over the final pellet for 30 minutes. For insemination, 50,000 motile sperm were added in 30 LL. In the presence of a male factor, a modified sperm preparation was used7 and 500,000 motile sperm were added to each tube.8 In order to maximize the number of sperm available in the male factor cases, over the last 6 months of the program, one to two additional semen collections were processed over the 6 days preceding laparoscopy and kept at room temperature under 5% C02 9 Next, 250,000 motile sperm were added to each oocyte from each of the fresh and incubated specimens. Modified Ham's F-10 medium containing 8.0 mm potassium10 was made up weekly using NaHC0 3, KHCOa, MgS0 4 7H20, and high-pressure liquid chromatography grade water from Mallinkrodt (Paris, KY). Streptomycin was added only to the semen wash and cervix wash. Only disposable culture ware (Falcon, Oxnard, CA) was used by adding an amount of water previously calculated to be required when added to the solutes. Media were sterilized using Nalgene 500-ml 0.2 Lm filtration units. Each batch was tested with 2-cell mouse embryos and was required to support development of 75% of embryos to blastocyst over 72 hours. Throughout the culture, 15% human fetal cord serum (FCS) was used as a serum supplement,10 each individually tested prior to use11 and required to support better mouse embryo growth than the corresponding control medium without a protein supplement (48% of sera were rejected). Culture was carried out in a humidified atmosphere of 5% C02 in room air. The occurrence of fertilization was examined at 15 to 18 hours following insemination by removal of the cumulus and corona by dissection with 26- gauge disposable needles in an organ culture dish (Falcon #3037). If more than two pronuclei were visualized, that conceptus was not transferred. If two pronuclei were not seen, insemination was repeated using the same sperm preparation that had been incubated overnight at 37 C, under 5% C02 Cleavage then was evaluated only immediately prior to transfer at 48 to 50 hours after oocyte retrieval. Embryos were chosen for transfer based on more rapid cleavage, absence of fragmentation, and even size of blastomeres. Over the first 12 months, up to six embryos were transferred; subsequently, the recommended limit was four. The excess embryos were cryopreserved over the last 3 months, using the method reported by Trounson and MohrP Transfer was performed in lithotomy and 20- to 30-degree Trendelenberg positions. Doxycycline (100 mg) was given orally on the morning of transfer and diazepam (10 mg) was administered 1 hour before the procedure. The embryos were placed in a single culture dish in 15% FCS in the C02-controlled isolette immediately adjacent to the patient. 5 After washing the cervix with unequilibrated Ham's F-10 medium containing both penicillin and streptomycin, the embryos were loaded into a prerinsed tomcat catheter (Monoject, St. Louis, M0)13 previously bent in the package to conform to the curve of that patient's cervical canal as determined prior to the treatment cycle. Using a Teflon-tipped, air-tight glass microliter syringe (Hamilton Co., Reno, NV), the catheter was loaded almost to the syringe tip, then removed and the syringe was pushed to zero and replaced. During 1984, the embryos were loaded by one of two methods. Method A Method A (January to June, 1984) was as follows: 10 Ll of 5% C02 in air, 10 LL of medium with the embryos one-third of the way along this column from the catheter tip, followed by 10 LL of 5% C02 in air and a 1-LL plug of medium at the catheter tip. Method B Method B (after June 1984) was as follows: 1 LL of 5% C02 in air, 15 to 25 LL of medium with the embryos one-third of the way along the column from the catheter tip, followed by 1 to 2 LL of 5% C02 in air and a 1-LL plug. The catheter was then inserted to 0.5 em from the fundus, measured by a 88 Meldrum et al. Evolution of an IVF-ET program Fertility and Sterility

4 disposable ruler used to measure the portion of the catheter remaining outside of the uterus. The transfer volume was then gently expelled and, after about 1 minute, the catheter was withdrawn with a 90-degree turn and checked for retained embryos. The patient then rested in a prone/trendelenberg position for 3 or 6 hours (alternated). If a retained embryo was noted, the transfer was repeated in 30 minutes. In one woman, an embryo was again retained, which was replaced after an additional 3 hours. The patients then rested at home for 48 hours and avoided prostaglandin-synthetase inhibitors, antihistimines, and intercourse until determination of the concentration of,8-hcg at 15 to 16 days following oocyte retrieval (day 0). Intramuscular progesterone in oil (25 mg) was injected daily, beginning on the day of transfer, until a negative,8-hcg or 28 days from transfer.1 A clinical pregnancy was defined as the finding of a gestational sac or the occurrence of a spontaneous abortion at least 28 days from transfer. A biochemical pregnancy was defined as a rise of the,8-hcg level to at least 10 miu/ml at 15 to 16 days from laparoscopy. Most patients were able to stop smoking14 during the treatment cycle and pregnancy. Recently, abstention from caffeine was also suggested.15 Most couples also abstained from coitus during the first 12 weeks of pregnancy. RESULTS Sixty-four cycles (29%) were bypassed due to an inadequate follicle or E2 response (16%), E2 drop during hmg (8%), ovulation (2%), excessive stimulation (1%), or an inaccessible ovary (2%). At least one oocyte was retrieved in 153 cycles (99.4%) with an average of ±8.9 oocytes harvested per laparoscopy. Of these oocytes, 77% were considered rnature, 17% intermediate, and 6% were immature. Sixty-two oocytes (5.9%) were clearly atretic. The fertilization rate in the nonmale factor, hmg only cycles was 60% of oocytes. In cycles in which a male factor was present, 41% of oocytes fertilized. More than two pronuclei were observed in 4.8% of all oocytes and 8% of fertilized oocytes. Fertilization rates for mature and intermediate oocytes were 67% and 44%, respectively. Of a subset of immature oocytes that were not transferred and further observed for 24 hours to evaluate cleavage, only 1 of 14 (7%) cleaved. Of oocytes with two pronuclei, 91.3% cleaved normally. A fractured zona was rarely seen ( <1% of oocytes). A total of 39 oocytes were retrieved from cul-de-sac fluid following oocyte aspiration, of which 28 (72%) fertilized and 24 (86%) cleaved. Transfer was carried out in 141 (92%) of the cycles, with an average of 3.4 embryos per transfer. During 1985 and 1986, when the transfer was limited to 4 embryos, 1.5 additional embryos formed per patient (3.2 per patient having extra embryos). Over the 3-month period from December 20, 1985 to March 20, 1986, 42 embryos were frozen on 12 patients. Thirty-three embryos were thawed and two clinical pregnancies (three clinical implantations, 9% of embryos thawed) were achieved (20% of patients, 17% of transfers) by transfer in normal cycles using daily LH and pelvic ultrasound monitoring.1 2 Table 1 shows the rates of total and clinical pregnancy by 6-month periods. A rapid improvement over the first year was noted, with the clinical pregnancy rate over the last 18 months being 34% and, over the entire 2! years, 31%. An additional two pregnancies (term deliveries) resulted from the 14 attempts (14%) at ultrasound-directed oocyte retrieval. Of the 4 7 clinical pregnancies that are now at least 18 weeks from conception, 9 (19%) Table 1 Total and Clinical Pregnancy Rates During Each 6-Month Period Since Program's Inception Clinical Laparoscopies Pregnancies pregnancies (%) (%) Sept 20, 1983-Mar 20, (12) 2 (12) Mar 20, 1984-Sept 20, (32) 5 (26) Sept 20, 1984-Mar 20, (37) 9 (30) Mar 20, 1985-Sept 20, (40) 13 (37) Sept 20, 1985-Mar 20, (38) 184 (34) 53 Total (34) 47 (31) a Two pregnancies resulted from later transfer of cryopreserved embryos. Vol. 48, No. 1, July 1987 Meldrum et al. Evolution of an IVF-ET program 89

5 Table 2 Pregnancy Rate According to the Day of hmg Administration on Which hcg Was Given in hmg-only Cycles Day of hmg Number Pregnant % Total have aborted. Thirty-seven women have delivered 51 healthy babies. No ectopic pregnancies have oc curred. The clinical pregnancy rates with the var ious infertility factors were: tubal, 28% ; endome triosis, 39%; idiopathic, 14%; and male factor, 32%. In the women with a history of endometriosis, a similar rate of pregnancy occurred whether there was no active endometriosis observed at the time of oocyte aspiration (7 /14) or whether endometriosis was cauterized (3/12) or not cauterized (3/9). More than one gestational sac was observed in 22 (47%) of the clinical pregnancies, 6 of which (27%) evolved into singleton gestations. All but two of the multiple pregnancies were twins. A triplet and quadruplet pregnancy resulted from the transfer of five and six embryos, respectively, and both deliv ered healthy babies. The rate of spontaneous abor tion was not different in the women with multiple implantations (18%) compared with those with a single implantation. A high proportion of all clini cal pregnancies (55%) experienced bleeding during the first few weeks of gestation. Table 2 shows the rate of clinical pregnancy ac cording to the day of medication when hcg was given. A similar rate of clinical pregnancy was noted regardless of the duration of hmg adminis tration, which varied from 6 to 10 days. The mean E 2 level on the day of hcg was and 1518 pg/ml with the hmg-only and the CC hmg regimens, respectively. The implantation rate (number of gestational sacs per embryo re placed) per embryo with the combination of CC and hmg was much lower (4%) when the E 2 on the Table 3 day of hcg was < 1000 pg/ml than when it ex ceeded 1000 pg/ml (20% ), whereas with hmg only, the implantation rates below and above this level (13% and 17%, respectively) were similar. Table 3 shows the rate of clinical pregnancy and the implantation rate per embryo with the two methods of loading the transfer catheter during 1984 when the remaining techniques remained quite constant. A significant (P < 0.05) increase was observed in the number of clinical implanta tions, with the rate of implantation per embryo increasing from 5.5% to 19.4% in association with the use of less air and a lower total volume. Table 4 shows the rate of clinical pregnancy versus the uterine depth. No difference was noted in the rate of successful implantation with uteri varying from 6 to >9 em. Of the three women with uterine myomata of 8 weeks' size or greater gesta tional size, one conceived and delivered a full-term pregnancy. No difference was noted with 3 versus 6 hours of bed rest or whether the uterus was ante rior or posterior. Six of 1 1 women needing repeat transfer had clinical pregnancies. Conception and delivery occurred in the one woman having three ET attempts. DISCUSSION In establishing an IVF-ET program, it is ex tremely important to choose only techniques that have been used by other successful programs. Our results illustrate the value of combining what we considered were the strong points of a number of successful teams rather than following one pro gram in all respects. We chose the high-dose hmg regimen1 for ovar ian stimulation in order to avoid potential adverse effects of CC on the endometrium. More recently, Rogers et ap6 have calculated a higher uterine re ceptivity with use of hmg-only, compared with CC-hMG cycles. To examine this question, we controlled for number of embryos replaced by cal- Clinical Implantations Occurring in Association with Two Methods of Loading of the Tomcat Catheter No. of transfers Cleaving embryos Clinical pregnancies per transfer A 19 B Clinical implantations Implantations per embryo 3 18" % p 90 < % Meldrum et al. Evolution of an IVF-ET program Fertility and Sterility

6 Table 4 Clinical Pregnancies Occurring in Association with Different Uterine Depths and Positions in 142 Cycles in Which Embryo Transfer Was Done Total 28 Pregnant 8 % 29 Depth em Position Anterior Posterior culating the implantation rate per embryo transferred. Our finding of a very low implantation rate per embryo in CC-hMG cycles with a low serum E 2 on the day of hcg injection is consistent with an antiestrogenic effect of the medication. The high-dose hmg regimen and the aspiration techniques that we used resulted in a high number of oocytes retrieved per laparoscopy. The fertilization and cleavage rates were consistent with the number of oocytes recovered.17 The correspondingly high mean number of embryos transferred per patient played a major part in the high pregnancy rate achieved, both through transfer in the IVF cycle and in later cycles after cryopreservation. Considering both contributions to pregnancy, the rate of clinical pregnancy per laparoscopy was 31%. Extrapolating these results, it may be anticipated that an additional 5% to 10% will be achieved in the total rate of pregnancy per laparoscopy when all extra embryos are frozen and transferred. We have considered the initial stimulation as a trial because of reports4 17 of a markedly reduced pregnancy rate when the serum E 2 falls during hmg or when a low E 2 response is achieved. However, our use of CC-hMG as the alternate regimen for poor responders yielded a very low implantation rate. Sequential increase of the hmg dose in subsequent cycles has recently been shown to be a highly effective treatment for low responders.18 This observation is supported by a recent finding of a significantly lower circulating level of FSH in poor responders, 19 presumably due to altered metabolism of injected hmg. We used both follicle diameter and serum E 2 to evaluate oocyte maturity because of the inherent variability of either index alone. Follicle measurements vary because of a number of technical factors20 and circulating estrogen is a variable reflection of estrogen production, depending on the metabolic clearance rate of the individual woman. The similar rate of pregnancy regardless of the duration of hmg therapy (Table 2) supports the use of these indices to individualize the timing of hcg injection according to the optimum point of follicle maturation. The lower rate of fertilization per oocyte in this series was presumably related to the high number of oocytes retrievedp Because of the strong hmg stimulation employed, it is likely that oocytes that would have been classified as immature following lower doses of hmg underwent cumulus expansion with the higher hmg dose. The immature oocytes that we retrieved, generally from very small follicles, fertilized and cleaved at a very low rate. Oocytes retrieved from the cul-de-sac fertilized and cleaved at a normal rate. Since the ph of the cul-de-sac fluid would have remained unchanged with use of a 5% C0 2 pneumoperitoneum, these 24 resulting embryos can be assumed to have contributed to the occurrence of pregnancy in some of our patients. We chose media components from manufacturers used by other successful programs, a highly pure commercial water, 1 and used only disposable materials for media preparation. These measures may help to. prevent introduction of traces of embryotoxic materials, which probably explains the marked variation of media quality demonstrated by a common testing facility.21 The addition of 5 mm potassium22 and human FCS10 have both been shown to result in a significantly higher pregnancy rate. Since some human cord sera have a deleterious effect on mouse embryo development, 11 each was individually tested and only those testing superior to media alone were used. The rate of clinical abortion has been higher with IVF (30%) than with spontaneous pregnancy.23 The high rates of multiple pregnancy, resorption of sacs, and early pregnancy bleeding indicate that multiple implantations may play a role in creating a less stable intrauterine environment. Probably due to the small sample size, we were not able to confirm an increased rate of abortion with multiple implantations. By excluding multipronucleate oocytes from transfer, the rate of spontaneous abortion could be reduced by as much as 25%, because one-quarter of abortuses are polyploid.24 This, together with abstention from smoking, caffeine, and coitus, may have helped to limit our rate of fetal loss (19%) to a rate similar to that observed for women with infertility who conceive with standard treatments. Vol. 48, No. 1, July 1987 Meldrum et al. Evolution of an IVF-ET program 91

7 Improvement of the clinical pregnancy rate during the evolution of our program was most strikingly associated with a reduction of total transfer volume and the proportion of air. In the lithotomy position, air and/or excess volume may displace the embryos down into the lower uterine segment, cervix, or even into the vagina. 2 5 We observed the same pregnancy rate (8 of 28 transfers) in women with uterine depths < 7 em as in those with larger cavities. This was significantly different (P < 0.01, Fisher exact test) from the experience of Sher et ap8 (0 of 31 transfers) using a 40- to 90-.tl transfer volume. These results sugg st that minor variations of transfer technique may impact on the chance of implantation. Because low fundal implantation is a common finding in pregnancies that eventually abort, lack of consistent high fundal placement of embryos may be partly responsible for the higher rate of spontaneous abortion generally observed with IVF-ET. 2 3 The high rate of multiple pregnancy that we observed suggests a high level of embryo quality.16 It may also be a function of transfer volume, since the rate of multiple implantation would be expected to be reduced by that proportion of transfer fluid that is not retained in the uterine fundus. As these factors are refined by a particular IVF program, it is necessary to further limit the number of embryos transferred, with the excess embryos being cryopreserved for transfer in other cycles. In summary, our IVF-ET program has achieved a high rate of success by combining aspects of technique introduced by other successful teams. We noted a marked increase in our rate of pregnancy associated with refinement of transfer methods, with clinical pregnancy occurring in 34% of laparoscopies over the last 18 months. We also have attributed our high rate of success to a strong ovarian, hmg-only stimulation, individualization of stimulation and hcg administration, use of 5% C02 pneumoperitoneum, a high oocyte retrieval rate, handling of oocytes and embryos in a C02- controlled pediatric isolette, rigid quality control of media, supplemented with individually tested human fetal cord sera, atraumatic, high fundal transfer of embryos, and the transfer of cryopreserved embryos in subsequent normal cycles. Acknowledgments. We appreciate the dedicated care of Denise Randle, R.N., and the excellent technical assistance of Tracey Moreno, B.S., and Villa Azrhmer, B.S. REFERENCES 1. 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8 ,, pregnancy and association with late spontaneous abortion. Am J Obstet Gynecol 154:14, Rogers PAW, Milne BJ, Trounson AO: A model to show human uterine receptivity and embryo viability following ovarian stimulation for in vitro fertilization. J In Vitro Fert Embryo Transfer 3:93, Lopata A: Concepts in human in vitro fertilization and embryo transfer. Fertil Steril 40:289, Sher G, Knutzen V, Stratton C, Chotiner H, Mayville J: In vitro fertilizatibn and embryo transfer: two-year experience. Obstet Gynecol 67:309, Ben-Rafael Z, Strauss JF III, Mastroianni L Jr, Flickinger GL: Differences in ovarian stimulation in human menopausal gonadotropin treated woman may be related to follicle-stimulating hormone accumulation. Fertil Steril 46:586, Prins GS, Vogelzang RL: Inherent sources of ultrasound variability in relation to follicular measurements. J In Vitro Fert Embryo Transfer 1:221, Ackerman SB, Stokes GL, Swanson RJ, Taylor SP, Fenwick L: Toxicity testing for human in vitro fertilization programs. J In Vitro Fert Embryo Transfer 2:132, Quinn P, Kerin JF, Warnes GM: Improved pregnancy rate in human in vitro fertilization with the use of a medium based on the composition of human tubal fluid. Fertil Steril 44:493, Seppala M: The world collaborative report on in vitro fertilization and embryo replacement: current state of the art in January Ann NY Acad Sci 442:558, Boue J, Boue A: Les avortements spontanes humains. Etudes cytogenetiques et epidemiologiques. Rev Fr Gynecol Obstet 68:625, Poindexter AN III, Thompson DJ, Gibbons WE, Findley WE, Dodson MG, Young RL: Residual embryos in failed embryo transfer. Fertil Steril 46:262, 1986 Vol. 48, No. 1, July 1987 Meldrum et al. Evolution of an IVF-ET program 93