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Evolution of a highly successful in vitro fertilization-embryo transfer program
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 , 1987. * Reprint requests: David R. Meldrum, M.D., In Vitro Fertil ization Center, AMI South Bay Hospital, 514 North Prospect Avenue, Redondo Beach, California 90277. 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.
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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
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 ap propriate treatment was given. Routine semen cul ture was not done. Ovarian stimulation was initiated by injection of 225 IU of human menopausal gonadotropins (hMG, Pergonal, Serono Laboratories Inc., Ran dolph, MA) daily starting on day 2 or 3 of the men strual 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 ci trate (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 ultra sound 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 promi nent 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 gonado tropin injection and hCG was kept to no more than 28 hours and generally to 14 hours. 3 Patients re sponding to stimulation with only a single mature follicle or with a peak estradiol (E 2 ) 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 E 2 levels at 9 A.M. (Immune-Estradiol P 25, Pantex, Vol. 48, No. 1 , July 1987
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 1 1 A.M.). In some cases, if the level of E 2 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 E 2 con centration 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 E 2 sample was always run simulta neously and, if any drop was observed prior to hCG, the cycle was bypassed. 4 No cycles were cancelled based on an E 2 drop after hCG. Thirty to 60 min utes before laparoscopy, pelvic ultrasound was re peated. The cycle was cancelled if ovulation was detected, unless only the largest follicle had col lapsed, 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 bal anced anesthesia, which was initiated after prepar ing the patient. Pneumoperitoneum was estab lished with 5% C0 2 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 14gauge Teflon catheter through a needle trocar placed 6 em above the symphysis in the midline. Oocytes were collected into prewarmed 15-ml Fal con tubes (#2057, Falcon Plastics, Oxnard, CA) through a Silicone bung at 100 mm Hg negative pressure. Follicles were flushed 2 to 5 times, de pending on the number of follicles, with equili brated Ham's F-10 medium (Flow Laboratories, McClean, VA) containing 40 U/ml of sodium hepa rin. 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 con trolled, humidified, pediatric isolette at 3 7 ° 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
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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 (Fal con #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 ejacu lation 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 mil liliter of 2.5 ml of medium that had been layered over the final pellet for 30 minutes. For insemina tion, 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% C0 2 • 9 Next, 250,000 motile sperm were added to each oocyte from each of the fresh and incubated specimens. Modified Ham's F - 1 0 medium containing 8.0 mM potassium10 was made up weekly using NaHC0 3 , KHCO a , MgS0 4 7H 2 0, and high-pres sure liquid chromatography grade water from Mal linkrodt (Paris, KY) . Streptomycin was added only to the semen wash and cervix wash. Only dispos able 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 filtra tion 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% C0 2 in room air. The occurrence of fertilization was examined at 15 to 18 hours following inse mination by removal of the cumulus and corona by dissection with 26•
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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 re peated using the same sperm preparation that had been incubated overnight at 37°C, under 5% C0 2 • Cleavage then was evaluated only immediately prior to transfer at 48 to 50 hours after oocyte re trieval. 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 em bryos 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 ( 1 00 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 C0 2 -controlled isolette immediately adjacent to the patient. 5 After washing the cervix with unequili brated Ham's F-10 medium containing both peni cillin and streptomycin, the embryos were loaded into a prerinsed tomcat catheter (Monoject, St. Louis, M0) 1 3 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 fol lows: 10 �Ll of 5% C0 2 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% C0 2 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% C0 2 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% C0 2 in air and a 1-�LL plug. The catheter was then inserted to 0.5 em from the fundus, measured by a
Fertility and Sterility
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 re tained, which was replaced after an additional 3 hours. The patients then rested at home for 48 hours and avoided prostaglandin-synthetase inhib itors, antihistimines, and intercourse until deter mination of the concentration of ,8-hCG at 15 to 16 days following oocyte retrieval (day 0). Intramus cular 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 sponta neous 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 preg nancy. 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 E 2 response (16% ), E 2 drop during hMG (8% ) , ovulation (2%), excessive stimu lation ( 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 laparos copy. Of these oocytes, 77% were considered rnaTable 1
ture, 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 oo cytes 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 follow ing 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 em bryos 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 im plantations, 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 improve ment 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%)
Total and Clinical Pregnancy Rates During Each 6-Month Period Since Program's Inception Pregnancies
Laparoscopies
(%)
(%)
Sept 20, 1983-Mar Mar 20, 1984-Sept Sept 20, 1984-Mar Mar 20, 1985-Sept Sept 20, 1985-Mar Total a
20, 20, 20, 20, 20,
1984 1984 1985 1985 1986
17 19 30 35 53
2 6 11 14 204
154
Clinical pregnancies
(12) (32) (37) (40) (38)
53 (34)
2 5 9 13 184
(12) (26) (30) (37) (34)
47 (31)
Two pregnancies resulted from later transfer of cryopreserved embryos.
Vol. 48, No. 1, July 1987
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Table 2 Pregnancy Rate According to the Day of hMG Administration on Which hCG Was Given in hMG-only Cycles
Day of hMG 6 18 6 33
Number Pregnant %
7 45 10 22
8 39 15 38
9 16 5 31
10 1 1 100
Total 119 37 31
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 1 128 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 28
55 93
16 32
Clinical implantations
Implantations per embryo
3 18"
5.5 19.4
%
•p
90
<
%
0.05.
Meldrum et al.
Evolution of an IVF-ET program
Fertility and Sterility
Table 4 Clinical Pregnancies Occurring in Association with Different Uterine Depths and Positions in 142 Cycles in Which Embryo Transfer Was Done
Depth Position
em
6.0-6.75 7.0-7.75 8.0-8.75 Total Pregnant %
28 8 29
68 24 35
35 10 29
9 11 3 27
Anterior Posterior 121 40 33
21 5 24
culating the implantation rate per embryo trans ferred. 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 fertiliza tion and cleavage rates were consistent with the number of oocytes recovered.17 The correspond ingly high mean number of embryos transferred per patient played a major part in the high preg nancy 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 3 1 % . Extrapolating these results, it may b e 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. How ever, our use of CC-hMG as the alternate regimen for poor responders yielded a very low implanta tion rate. Sequential increase of the hMG dose in subsequent cycles has recently been shown to be a highly effective treatment for low responders. 1 8 This observation is supported by a recent finding of a significantly lower circulating level of FSH in poor responders, 1 9 presumably due to altered me tabolism 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 measure ments vary because of a number of technical fac tors20 and circulating estrogen is a variable reflec tion of estrogen production, depending on the met abolic clearance rate of the individual woman. The Vol. 48, No. 1, July 1987
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 matura tion. 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 folli cles, 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 contrib uted to the occurrence of pregnancy in some of our patients. We chose media components from manufac turers 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 em bryotoxic materials, which probably explains the marked variation of media quality demonstrated by a common testing facility. 2 1 The addition of 5 mM potassium 22 and human FCS10 have both been shown to result in a significantly higher pregnancy rate. Since some human cord sera have a deleteri ous effect on mouse embryo development, 11 each was individually tested and only those testing supe rior 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 oo cytes from transfer, the rate of spontaneous abor tion could be reduced by as much as 25% , because one-quarter of abortuses are polyploid. 24 This, to gether 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.
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91
Improvement of the clinical pregnancy rate dur ing the evolution of our program was most strik ingly 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, cer vix, 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 aP 8 (0 of 31 transfers) using a 40- to 90-�.tl transfer volume. These results sugg�st that minor varia tions of transfer technique may impact on the chance of implantation. Because low fundal im plantation 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 gener ally observed with IVF-ET. 23 The high rate of multiple pregnancy that we ob served suggests a high level of embryo quality. 1 6 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 fac tors are refined by a particular IVF program, it is necessary to further limit the number of embryos transferred, with the excess embryos being cryo preserved for transfer in other cycles. In summary, our IVF-ET program has achieved a high rate of success by combining aspects of tech nique 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 laparo scopies over the last 18 months. We also have at tributed our high rate of success to a strong ovar ian, hMG-only stimulation, individualization of stimulation and hCG administration, use of 5 % C0 2 pneumoperitoneum, a high oocyte retrieval rate, handling of oocytes and embryos in a C0 2 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 cryopre served 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.
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pregnancy and association with late spontaneous abortion. Am J Obstet Gynecol 154:14, 1986 16. 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, 1986
21.
22.
17. Lopata A: Concepts in human in vitro fertilization and em bryo transfer. Fertil Steril 40:289, 1983 18. Sher G, Knutzen V, Stratton C, Chotiner H, Mayville J: In vitro fertilizatibn and embryo transfer: two-year experience. Obstet Gynecol 67:309, 1986 19. Ben-Rafael Z, Strauss JF III, Mastroianni L Jr, Flickinger GL: Differences in ovarian stimulation in human meno pausal gonadotropin treated woman may be related to folli cle-stimulating hormone accumulation. Fertil Steril 46:586, 1986
23.
24.
25.
, ,
Evolution of an IVF-ET program
93
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 , 1987. * Reprint requests: David R. Meldrum, M.D., In Vitro Fertil ization Center, AMI South Bay Hospital, 514 North Prospect Avenue, Redondo Beach, California 90277. 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.
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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
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 ap propriate treatment was given. Routine semen cul ture was not done. Ovarian stimulation was initiated by injection of 225 IU of human menopausal gonadotropins (hMG, Pergonal, Serono Laboratories Inc., Ran dolph, MA) daily starting on day 2 or 3 of the men strual 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 ci trate (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 ultra sound 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 promi nent 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 gonado tropin injection and hCG was kept to no more than 28 hours and generally to 14 hours. 3 Patients re sponding to stimulation with only a single mature follicle or with a peak estradiol (E 2 ) 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 E 2 levels at 9 A.M. (Immune-Estradiol P 25, Pantex, Vol. 48, No. 1 , July 1987
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 1 1 A.M.). In some cases, if the level of E 2 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 E 2 con centration 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 E 2 sample was always run simulta neously and, if any drop was observed prior to hCG, the cycle was bypassed. 4 No cycles were cancelled based on an E 2 drop after hCG. Thirty to 60 min utes before laparoscopy, pelvic ultrasound was re peated. The cycle was cancelled if ovulation was detected, unless only the largest follicle had col lapsed, 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 bal anced anesthesia, which was initiated after prepar ing the patient. Pneumoperitoneum was estab lished with 5% C0 2 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 14gauge Teflon catheter through a needle trocar placed 6 em above the symphysis in the midline. Oocytes were collected into prewarmed 15-ml Fal con tubes (#2057, Falcon Plastics, Oxnard, CA) through a Silicone bung at 100 mm Hg negative pressure. Follicles were flushed 2 to 5 times, de pending on the number of follicles, with equili brated Ham's F-10 medium (Flow Laboratories, McClean, VA) containing 40 U/ml of sodium hepa rin. 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 con trolled, humidified, pediatric isolette at 3 7 ° 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
Meldrum et al.
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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 (Fal con #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 ejacu lation 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 mil liliter of 2.5 ml of medium that had been layered over the final pellet for 30 minutes. For insemina tion, 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% C0 2 • 9 Next, 250,000 motile sperm were added to each oocyte from each of the fresh and incubated specimens. Modified Ham's F - 1 0 medium containing 8.0 mM potassium10 was made up weekly using NaHC0 3 , KHCO a , MgS0 4 7H 2 0, and high-pres sure liquid chromatography grade water from Mal linkrodt (Paris, KY) . Streptomycin was added only to the semen wash and cervix wash. Only dispos able 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 filtra tion 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% C0 2 in room air. The occurrence of fertilization was examined at 15 to 18 hours following inse mination by removal of the cumulus and corona by dissection with 26•
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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 re peated using the same sperm preparation that had been incubated overnight at 37°C, under 5% C0 2 • Cleavage then was evaluated only immediately prior to transfer at 48 to 50 hours after oocyte re trieval. 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 em bryos 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 ( 1 00 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 C0 2 -controlled isolette immediately adjacent to the patient. 5 After washing the cervix with unequili brated Ham's F-10 medium containing both peni cillin and streptomycin, the embryos were loaded into a prerinsed tomcat catheter (Monoject, St. Louis, M0) 1 3 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 fol lows: 10 �Ll of 5% C0 2 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% C0 2 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% C0 2 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% C0 2 in air and a 1-�LL plug. The catheter was then inserted to 0.5 em from the fundus, measured by a
Fertility and Sterility
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 re tained, which was replaced after an additional 3 hours. The patients then rested at home for 48 hours and avoided prostaglandin-synthetase inhib itors, antihistimines, and intercourse until deter mination of the concentration of ,8-hCG at 15 to 16 days following oocyte retrieval (day 0). Intramus cular 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 sponta neous 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 preg nancy. 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 E 2 response (16% ), E 2 drop during hMG (8% ) , ovulation (2%), excessive stimu lation ( 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 laparos copy. Of these oocytes, 77% were considered rnaTable 1
ture, 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 oo cytes 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 follow ing 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 em bryos 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 im plantations, 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 improve ment 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%)
Total and Clinical Pregnancy Rates During Each 6-Month Period Since Program's Inception Pregnancies
Laparoscopies
(%)
(%)
Sept 20, 1983-Mar Mar 20, 1984-Sept Sept 20, 1984-Mar Mar 20, 1985-Sept Sept 20, 1985-Mar Total a
20, 20, 20, 20, 20,
1984 1984 1985 1985 1986
17 19 30 35 53
2 6 11 14 204
154
Clinical pregnancies
(12) (32) (37) (40) (38)
53 (34)
2 5 9 13 184
(12) (26) (30) (37) (34)
47 (31)
Two pregnancies resulted from later transfer of cryopreserved embryos.
Vol. 48, No. 1, July 1987
Meldrum et al.
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Table 2 Pregnancy Rate According to the Day of hMG Administration on Which hCG Was Given in hMG-only Cycles
Day of hMG 6 18 6 33
Number Pregnant %
7 45 10 22
8 39 15 38
9 16 5 31
10 1 1 100
Total 119 37 31
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 1 128 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 28
55 93
16 32
Clinical implantations
Implantations per embryo
3 18"
5.5 19.4
%
•p
90
<
%
0.05.
Meldrum et al.
Evolution of an IVF-ET program
Fertility and Sterility
Table 4 Clinical Pregnancies Occurring in Association with Different Uterine Depths and Positions in 142 Cycles in Which Embryo Transfer Was Done
Depth Position
em
6.0-6.75 7.0-7.75 8.0-8.75 Total Pregnant %
28 8 29
68 24 35
35 10 29
9 11 3 27
Anterior Posterior 121 40 33
21 5 24
culating the implantation rate per embryo trans ferred. 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 fertiliza tion and cleavage rates were consistent with the number of oocytes recovered.17 The correspond ingly high mean number of embryos transferred per patient played a major part in the high preg nancy 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 3 1 % . Extrapolating these results, it may b e 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. How ever, our use of CC-hMG as the alternate regimen for poor responders yielded a very low implanta tion rate. Sequential increase of the hMG dose in subsequent cycles has recently been shown to be a highly effective treatment for low responders. 1 8 This observation is supported by a recent finding of a significantly lower circulating level of FSH in poor responders, 1 9 presumably due to altered me tabolism 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 measure ments vary because of a number of technical fac tors20 and circulating estrogen is a variable reflec tion of estrogen production, depending on the met abolic clearance rate of the individual woman. The Vol. 48, No. 1, July 1987
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 matura tion. 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 folli cles, 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 contrib uted to the occurrence of pregnancy in some of our patients. We chose media components from manufac turers 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 em bryotoxic materials, which probably explains the marked variation of media quality demonstrated by a common testing facility. 2 1 The addition of 5 mM potassium 22 and human FCS10 have both been shown to result in a significantly higher pregnancy rate. Since some human cord sera have a deleteri ous effect on mouse embryo development, 11 each was individually tested and only those testing supe rior 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 oo cytes from transfer, the rate of spontaneous abor tion could be reduced by as much as 25% , because one-quarter of abortuses are polyploid. 24 This, to gether 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.
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91
Improvement of the clinical pregnancy rate dur ing the evolution of our program was most strik ingly 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, cer vix, 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 aP 8 (0 of 31 transfers) using a 40- to 90-�.tl transfer volume. These results sugg�st that minor varia tions of transfer technique may impact on the chance of implantation. Because low fundal im plantation 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 gener ally observed with IVF-ET. 23 The high rate of multiple pregnancy that we ob served suggests a high level of embryo quality. 1 6 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 fac tors are refined by a particular IVF program, it is necessary to further limit the number of embryos transferred, with the excess embryos being cryo preserved for transfer in other cycles. In summary, our IVF-ET program has achieved a high rate of success by combining aspects of tech nique 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 laparo scopies over the last 18 months. We also have at tributed our high rate of success to a strong ovar ian, hMG-only stimulation, individualization of stimulation and hCG administration, use of 5 % C0 2 pneumoperitoneum, a high oocyte retrieval rate, handling of oocytes and embryos in a C0 2 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 cryopre served 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.
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REFERENCES
1. Laufer N, DeChemey AH, Haseltine FP, Polan ML, Mezer HC, Dlugi AM, Sweeney D, Nero F, Naftolin F: The use of high-dose human menopausal gonadotropin in an in vitro fertilization program. Fertil Steril 40:734, 1983 2. Cedars MI, Steingold KA, de Ziegler D, Chetkowski R, Hamilton F, Meldrum DR: Outcome of ovarian stimulation, in vitro fertilization and embryo transfer in oligomenorrheic women. Unpublished data 3. Laufer N, DeChemey AH, Tarlatzis BC, Zuckerman AL, Polan ML, Dlugi AM, Graebe R, Barnea ER, Naftolin F: Delaying human chorionic gonadotropin administration in human menopausal gonadotropin-induced cycles decreases successful in vitro fertilization of human oocytes. Fertil Steril 42: 198, 1984 4. Jones HW Jr, Acosta A, Andrews MC, Garcia JE, Jones GS, Mantzavinos T, McDowell J, Sandow B, Veeck L, Whibley T, Wilkes C, Wright G: The importance of the follicular phase to success and failure in in vitro fertilization. Fertil Steril 40:317, 1983 5. Chetkowski RJ, Nass TE, Matt DW, Hamilton F, Steingold KA, Randle D, Meldrum DR: Optimization of hydrogen ion concentration during aspiration of oocytes and culture and transfer of embryos. J In Vitro Fert Embryo Transfer 2:207, 1985 6. Veeck LL, Wortham JWE Jr, Witmyer J, Sandow BA, Acosta AA, Garcia JE, Jones GS, Jones HW Jr: Maturation and fertilization of morphologically immature human oo cytes in a program of in vitro fertilization. Fertil Steril 39:594, 1983 7. Mahadevari MM, Trounson AO, Leeton JF: The relation ship of tubal blockage, infertility of unknown cause, sus pected male infertility, and endometriosis to success of in vitro fertilization and embryo transfer. Fertil Steril 40:755, 1983 8. Diamond MP, Rogers BJ, Vaughn WK, Wentz AC: Effect of the number of inseminating sperm and the follicular stimu lation protocol on in vitro fertilization of human oocytes in male factor and non- male factor couples. Fertil Steril 44:499, 1985 9. Cohen J, Fehilly , CB, Walters DE: Prolonged storage of human spermatozoa at room temperature or in a refrigera tor. Fertil Steril 44:254, 1985 10. Leung PCS, Gronow MJ, Kellow GN, Lopata A, Speirs AL, McBain JC, du Plessis YP, Johnston 1: Serum supplement in human in vitro fertilization and embryo development. Fertil Steril 41:36, 1984 11. Cheung SW, Strickler RC, Yang VC, de Vera M, Spitznagal EL: A mouse embryo culture system for quality control testing of human in vitro fertilization and embryo transfer media and fetal cord sera. Gamete Res 1 1 :4 1 1 , 1985 12. Trounson A, Mohr L: Human pregnancy following cryopre servation, thawing and transfer of an 8-cell embryo. Nature 305:707, 1983 13. Quinn P, Warnes GM, Kerin JF, Kirby C: Culture factors in relation to the success of human in vitro fertilization and embryo transfer. Fertil Steril 41:202, 1984 14. Kline J, Stein ZA, Susser M, Warburton D: Smoking: a risk factor for spontaneous abortion. N Engl J Med 297:793, 1977 15. Srisuphan W, Bracken MB: Caffeine consumption during
Fertility and Sterility
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