The use of high-dose human menopausal gonadotropin in an in vitro fertilization program

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1 FERTILITY AND STERILITY Copyright 98 The American Fertility Society Vol. 40, No.6, December 98 Printed in U.8A. The use of high-dose human menopausal gonadotropin in an in vitro fertilization program Neri Laufer, M.D.*t Alan H. DeCherney, M.D. Florence P. Haseltine, Ph.D., M.D. Mary Lake Polan, Ph.D., M.D. Howard C. Mezer, M.D. Alexander M. Dlugi, M.D. Dorothy Sweeney, B.S. Filomena Nero, R.N. Frederick Naftolin, M.D., D.Phil. Department of Obstetrics and Gynecology, Yale University School of Medicine, New Haven, Connecticut Sixty-three normal ovulatory women suffering from obstructive tubal disease not corrected by previous surgery were enrolled in an in vitro fertilization (lvf) program. To achieve a large number of mature follicles, a relatively high dose of human menopausal gonadotropin (hmg) was administered (9 ± 4 ampules/cycle). Monitoring consisted of daily follicular ultrasonography and serum estradiol measurements. Human chorionic gonadotropin (0,000 IU) was administered when more than two large follicles (.6 to.8 cm in diameter) were visualized. Fifty-five laparoscopies for oocyte retrieval were performed. A mean of 4. follicles per woman were aspirated, and. oocytes per woman were recovered. The oocytes were preincubated for 8 or 4 hours according to the morphologic degree of mucification and dispersal of the oocyte-corona-cumulus complex. Seventy-seven percent of the oocytes were fertilized and were transferred into the uterus 8 to 40 hours after insemination. Fifty-two women received One to eight embryos (mean,.5 ±.9), and 9 (7%) conceived. This regimen of high-dose hmg precludes the need for serum or urine luteinizing hormone monitoring, because the occurrence of spontaneous ovulation is low. It is valuable in increasing the number of fertilizable oocytes, the percentage of women undergoing embryo transfer, and compensates with multiple oocyte transfer for the high embryonic loss involved in IVF. Fertil Steril 40:74, 98 Pregnancy rates in in vitro fertilization (IVF) programs may be improved if multiple embryos are transferred into the uterus. Trounson et al. and Jones et al. have reported a 0% pregnancy rate for women implanted with two embryos, compared with an 8% to % success rate for Received May 6, 98; revised and accepted August 9, 98. *Andrew W. Mellon Foundation Fellow in Reproductive Sciences on leave from the Department of Obstetrics and Gynecology, Hadassah Medical School, Jerusalem, Israel. treprint requests: Neri Laufer, M.D., Department of Obstetrics and Gynecology, Yale University School of Medicine, Cedar Street, New Haven, Connecticut women implanted with only one embryo. To initiate the growth of multiple follicles and increase the yield of aspirated oocytes, many groups involved in IVF have used clomiphene citrate (CC) or a combination of CC followed by low-dose human menopausal gonadotropin (hmg) therapy. CC, although capable of inducing the growth of multiple follicles, is associated with a 5% abortion rate, primarily due to corpus luteum insufficiency.4 In vitro, CC was shown to reduce progesterone (P) production in both human corpora lutea 5 and rat follicles 6 and to induce spontaneous maturation of rat oocytes. 6 The drug was also shown to decrease both fertilization and the development of mouse oocytes in vivo and in vitro.7 74 Laufer et al. Human menopausal gonadotropin in IVF Fertility and Sterility

2 Because these clinical and laboratory data suggest a direct and undesired effect of CC on both the ovary and the oocyte itself, we elected to use only hmg for the induction of ovulation in IVF patients. Jones et a. reported a pregnancy/laparoscopy rate of % with a regimen of low-dose hmg. In an attempt to maximize the pregnancy rate by implanting more developing embryos, higher doses of hmg were used to induce ovulation in this study. This work describes our program and its results. PATIENT SELECTION MATERIALS AND METHODS Between September 5, 98, and January 5, 98, 6 women were enrolled in the IVF program. Their ages ranged from 5 to 7 years (mean,.5), and all women participating suffered from primary or secondary infertility of to 0 years' duration (mean, 5.5) as a result of tubal disease, including both tubal occlusions and peritubal adhesions. The minimum criteria for inclusion in the program included the following: The women were 7 years of age or younger; at least one ovary was accessible, as proven by a laparotomy or laparoscopy within the last years; there had been at least one previous attempt at reconstructive tubal surgery; there was a normal semen analysis (> 0 x 0 6 cells/ml, > 40% normal morphology, > 50% good progressive motility); there was a normal uterine cavity (as evidenced by previous hysterosalpingography); and there was proven ovulation, either spontaneous or drug-induced, without hyperprolactinemia. All women were in good general health and were taking no other medication. OVULATION INDUCTION AND MONITORING SYSTEM The patients received three ampules of hmg (Pergonal, Serono Laboratories, Inc., Randolph, MA), 5 IUlday of follicle-stimulating hormone and luteinizing hormone (LH) from day of the cycle for 5 days. The dose was increased from day 8 in a stepwise manner: by one or two additional ampules per day for another to 4 days, depending on the individual's ovarian response. A range of 5 to ampules per cycle ofhmg (mean, 9 ± 4) was administered to these women. Monitoring consisted of daily serum 7-estradiol (E ) and ovarian ultrasonographic studies (Siemens PHO-Sonic B-Scanner, Santa Clara, CA) starting on day 8 of the cycle. No measurements of serum LH were performed. Human chorionic gonadotropin (hcg) (0,000 IU) was administered intramuscularly when at least two large follicles (.6 to.8 cm in diameter) were visualized. Ultrasonography, and not the E level, was considered a more definitive measurement of follicular developme~t. This observation is based on former experiences and data collected from these cycles showing that the value of E predicting the number of developing follicles in induced cycles is limited. 9 LAPAROSCOPY AND ASPIRATION OF FOLLICLES Laparoscopy was performed in the morning, 6 to 8 hours after hcg administration. Ovarian ultrasonography was performed 0 minutes to hour prior to the procedure; and if fewer than two adequate follicles were visualized, the procedure was abandoned. Patients were intubated only after the abdomen was prepared and draped. A pneumoperitoneum was induced with CO, and the oocytes were aspirated utilizing a three-puncture technique.o Patients were not in the lithotomy position; and the vagina, cervix, and uterus were not instrumented in any way. The needle used for the aspiration was -gauge, with an introducer modified from the TRU-cut biopsy needle (N-7-04 Travenol Laboratories, Deerfield, IL). The needle was washed prior to aspiration with heparinized ( mg/ml) medium devoid of serum. A 0-ml DeLee suction trap (Argyle Company, St. Louis, MO) was connected to the aspirating needle by an extension tube connected to a continuous wall suction at 00 mm Hg. The ovaries were grasped by the ovarian ligament for fixation, and the follicles were entered in an avascular area. An average of ml of follicular fluid was aspirated. When blood-tinged follicular fluid was obtained, an equal volume of warm heparinized medium was added to the trap. No attempt was made to flush the aspirated follicles. The collection traps were rushed to the laboratory in a Styrofoam container cushioned with prewarmed saline infusion bags. The laboratory was located 5 to 7 minutes' walking distance from the operating theatre. CULTURE MEDIA AND FERTILIZATION Modified Ham's F-0 medium was prepared weekly from a stock solution as described by Lo- Vol. 40, No.6, December 98 Laufer et ai. Human menopausal gonadotropin in IVF 75

3 Figure The morphology ofoeee in hmg-treated women. Three main types of oeee were identified: immature oeee (m), tight corona and cumulus of a few layers only; intermediate oeee (In), partly dispersed cumulus and corona; mature oeee (Ma), advanced cumulus and corona dispersal allowing the visualization of the zona pellucida (original magnification, x 00, Hoffman Interference Optics). pata et al.lo Water used in medium preparation was ultrapure high-pressure liquid chromatography grade (Mallinckrodt, Paris, KY), and the osmolarity was adjusted to 80 mosmil. After gassing with 5% CO, 5% O, and 90% N for 0 minutes, the ph was adjusted to 7.4. The insemination medium (M) was supplemented with 0% of the individual woman's heat-inactivated serum. Heparinized medium contained mg/ml heparin (GIBCO, Grand Island, NY) constituted the morning of the procedure. Growth medium (GM) was similarly prepared to contain 0% serum as suggested by Marrs.l Ova and embryos were cultured in ml medium in organ culture dishes (#07, Falcon Plastics, La Jolla, CA) without oil. Prior to being used, the medium was incubated overnight at 7.5 C in sealed jars after gassing with 5% CO, 5% O, and 90% N. The length of preincubation prior to insemination was determined individually for each oocyte. This decision depended on the degree of cumulus mucification and corona cell dispersion present. Three main types of oocyte-corona-cumulus complexes (OCCCs) could be identified: immature OCCCs, with a tight corona and cumulus of a few layers only; intermediate OCCCs, with a dispersed cumulus but only a partly dispersed corona; and mature OCCCs, with advanced dispersion allowing the visualization of the zona pellucida (Fig. ). Immature OCCCs were preincubated for 4 hours prior to insemination, and more mature OCCCs were preincubated for 6 to 8 hours before insemination. A semen specimen given by the husband hour before insemination was prepared with two wash76 Laufer et al. Human menopausal gonadotropin in IVF ings as described by Lopata et al. 0 Eggs were inseminated with x 06 motile sperm in 5 to 50 fl.l immediately after the wash. The same sperm preparation was incubated overnight in a separate organ culture dish and used to fertilize the immature OCCCs and to observe sperm viability. The ova were moved from M to GM 6 to 8 hours after insemination, having been denuded of excess corona ~ells during transfer by shearing, using hand-drawn pipettes. These denuded oocytes were examined for the formation of pronuclei. At 8 to 40 hours after insemination, the - to 4-cell embryos were implanted in the uterus utilizing an 8- to 0-gauge Teflon-tipped catheter with a side opening devised by H. Jones (manufactured by W. Kelly, Johns Hopkins Hospital Workshop). Embryos were aspirated with a 00fl.l syringe (Hamilton Company, Reno, NV) and placed in a center column of0 to 40 fl.l of medium separated by 5 fl.l of air from adjacent medium columns of 0 to 5 fl.l each. The total amount of air and culture medium injected did not exceed 80 fl.l. A metal introducer was passed to cm into the cervix, through which the catheter was threaded. Women were given the implants in the knee-chest position if the uterus was anteverted and in the dorsal lithotomy position if the uterus was retroverted. TREATMENT AND MONITORING AFTER IMPLANTATION Patients were hospitalized for 4 hours and kept at bed rest. P, 5 mg/day intramuscularly, was begun on the day of implantation and continued throughout the luteal phase. Blood was drawn every days after the hcg injection for ~-hcg titer, P, and E. Conception was detected by a rising ~-hcg titer and followed up to 0 to weeks of gestation. Ultrasonographic studies were done weekly until 0 to weeks' gestation and biweekly thereafter. METHODOLOGY FOR THE MOUSE EMBRYO DEVELOPMENT QUALITY CONTROL TEST OF THE MEDIUM B6DF I/J hybrid immature female mice (Jackson Laboratory, Bar Harbor, ME) at 5 to 6 weeks of age, weighing 0 to 5 gm, and mature 6- to 8-month-old B6DF I/J males of previously proven fertility were used to produce fertilized eggs. The animals were housed in air-conditioned quarters that were illuminated between 8:00 A.M. and Fertility and Sterility

4 Table. Results of 55 Laparoscopies Performed Between September 5, 98 and January 5, 98 No. of laparoscopies No. of follicles aspirated No. of oocytes recovered Fertilized oocytes Transfers Pregnancies Follicles/woman Oocytes/woman Oocytes recovered/follicle Fertilization/recovered oocyte Transferllaparoscopy Pregnancy/transfer Pregnancy/laparoscopy % 77% 94% 7% 6% 7:00 P.M., and peueted food and water were available. Superovulation of the immature mice was accomplished using 0 IU pregnant mare's serum gonadotropin (Gestyl, N. V. Organon, Oss, The Netherlands) injected at 4:00 P.M. (day ) followed by 5 IU hcg (Sigma Chemical Company, St. Louis, MO) 48 hours later (day ). Females were then caged with males and left overnight. Mating was confirmed by the presence of a vaginal plug on the following morning (day 4). The animals were killed by cervical dislocation at noon (0 hours after hcg injection). Oviducts were dissected out, and the ovulated eggs were released into 0.-ml drops of culture medium by incising the ampulla with a 7 -gauge needle. The cumulus cells were dispersed with hyaluronidase solution (0.% dissolved in culture medium) (Sigma, 400 IUlmg) by repeated pipetting through a narrow fire-polished pipette. Oocytes from individual animals were transferred to 0. to 0. ml of medium under paraffin oil (white, light, Mallinckrodt) in tissue culture dishes (Falcon # 007). Culture was carried out at 7 C in 5% CO, 5% O, and 90% N in the medium used for human IVF. The pronuclear stage oocytes were followed for 96 hours up to the blastocyst stage to ensure that the medium was able to support early embryo development. RESULTS QUALITY CONTROL OF MEDIA Quality control was based on two tests: mouse embryo culture and the "sperm survival test." At the beginning of the program every new fresh batch of medium was tested, and the development of mouse embryos to the blastocyst stage was as- sessed. For over 4 consecutive months the rates of cleavage were stable (85%). We consider this cleavage rate to be evidence that the media is of acceptable quality. Stock medium is now tested only once every month. Normal sperm incubated in M should maintain their original motility for at least 4 hours. Usually, specimens examined after 48 hours were as vigorous as the original. No reduction was observed in sperm viability after a 4-hour incubation period for any of the specimens in this series of couples. OVULATION INDUCTION Sixty-three women were included in this phase of the program. Three patients ovulated prior to laparoscopy; two patients had only one follicle and the cycle was abandoned; and three patients showed no signs of ovarian response in spite of a relatively high dose ofhmg. In the remaining 55 women, at the time of hcg administration, the mean serum E level was 945 ± 690 pg/ml (range, 0 to 7 pg/ml); the mean number of follicles visualized by ultrasonography was 5. ±.4 (range, to 9), and the mean follicular diameter was.7 ± 0.45 cm (range, 0.8 to. cm). IN VITRO FERTILIZATION The results of 55 patients undergoing laparoscopy are presented in Table. In two women the ovaries were not accessible because of adhesions that could not be dissected during laparoscopy. In another, no oocytes were found in the follicular fluid of two aspirated follicles. A mean of 4. follicles were aspirated per woman, from which a mean of. ova were recovered. The distribution of OCCC in this group of patients is given in Table. It is evident that most ova (9%) were derived from intermediate and mature OCCCs. The overall fertilization rate evidenced by two pronuclei (for immature OCCCs) and cleavage (for intermediate and mature OCCC-derived ova) was 77%; no statistically significant difference was seen between the 9% fertilization rate for immature OCCCs and the 68% fertilization rate for mature OCCCs. It should be noted, however, that ova derived from immature OCCCs were implanted in the uterus at the pronuclear stage, 6 to 8 hours after fertilization. Therefore, we cannot assess whether these ova continued to cleave in vivo. Table summarizes the data on the rela- Vol. 40, No.6, December 98 Laufer et al. Human menopausal gonadotropin in IVF 77

5 Table. The Relationship Between oeee Maturity and Fertilization OCCC mor- Total Fertilized phologic ma- no. of only (two turity eggs pronuclei) -cell Immature 6 5 Intermediate 88 6 Mature 76 Cleaved Not fer- % fertil- -4-cell 4-6-cell tilized izeda 9% % 5 68% Total ano significant difference among the three groups. tionship between the number of ova implanted and conceptions. In this series pregnancies occurred only when three or more fertilized ova were implanted. Individual data for the women in whom implantation resulted in a clinical pregnancy are summarized in Table 4. No specific pattern offertilized ova or degree of embryo development seemed to differentiate successful from failed implantation. Similarly, no relationship between the symmetry of cleaved embryos and successful pregnancies was noted. Pregnancy occurred with fertilized ova containing asymmetric as well as symmetric blastomeres (Fig. A and B). The most advanced pregnancy is currently at 7 weeks of gestation, and the most recent is at 0 weeks. Only one woman (patient ) experienced clinical mild ovarian hyperstimulation resulting in bilaterally enlarged ovaries, reaching a diameter of 0 cm by day 0 after hcg administration. No ascites or pleural effusion occurred, and with bed rest the syndrome gradually resolved over the following 0 days. DISCUSSION The first pregnancies obtained by IVF in women were achieved with oocytes from unstimulated ovaries. Because the yield of oocytes in the natural cycle is low and the monitoring involved is expensive and time-consuming, most groups involved in IVF now use stimulated cycles with CC or a combination ofcc/hmg. The use ofhmg for induction of ovulation seems to have advantages over CC because of the apparently lower abortion rate associated with hmg treatment and because animal studies have shown a direct undesired effect of CC on oocyte maturation, fertilization, and early embryonic development. 6 - B Mild ovarian stimulation with hmg for oocyte retrieval in IVF was pioneered by Steptoe and Edwards but later abandoned. Jones et a. have been the only group so far to use solely hmg/ % hcg for induction of ovulation in IVF patients. According to their series, it could not be determined whether hmg was indeed better than CC for ovarian stimulation. This group corroborated previous observations by Trounson et ai., l who showed that a 0% pregnancy rate was achieved when two embryos were transferred into the uterus after IVF. However, because of the relatively low dose ofhmg used by the Norfolk group, only 50% of the women that underwent embryo transfer were given two embryos. In order to obtain a larger population of patients in this category, we estimated that with a 0% decrease in the success rate of each successive step of the procedure, an ideal cycle with five large follicles visualized on ultrasonography would yield the optimal number of two cleaved oocytes. Using an individualized regimen utilizing relatively high doses of hmg, the objective of obtaining at least two cleaved oocytes has been attained in over 80% of the transferred patients. However, it should be noted that even with this relatively high-dose hmg regimen (mean, 9 ampules/cycle), 5 of 6 patients did not show a sufficient ovarian response, and the cycle had to be abandoned. The observation that only of 6 women ovulated prior to laparoscopy strengthens the as- Table. Relationship of the Number of Implanted Oocytes to Successful Pregnancies Total no. of oocytes No. of Pregnancies Chemical implanted women pregnancies a Total 5 9 achemical pregnancy is defined as a transient increase of serum l-hcg. 78 Laufer et al. Human menopausal gonadotropin in IVF Fertility and Sterility

6 Table 4. The Population of Transferred Embryos Resulting in Successful Pregnancies Case Total no. of oocytes b Total C 46 Pronuclear" Cleaved -cell cell > 4-cell Not fertilized 6 No. of ultrasound gestational sacs 0d almmature ova fertilized after 4 hours of preincubation and implanted at the pronuclear stage. b Aborted at 0 weeks of gestation. CEighty-seven percent were fertilized. done resorbed at 8 weeks. II!I i!, sumption that the monitoring of serum or urine LH is not necessary with this hmg schedule. This provides some decrease in the expense of the IVF program and offers some flexibility in the timing of the hcg administration in order to coincide with operating room availability. Oocyte aspiration and transfer can be performed in the morning hours, thus not imposing an additional burden of work on the IVF team. Comparing the regimen of hmg we use with that of Jones et al., it is evident that at the higher doses of hmg a larger number of oocytes were recovered, and therefore the percentage of women transferred increased (. versus.8 oocytes per laparoscopy and 9% versus 79% transfers per laparoscopy, respectively). Although our pregnancy/laparoscopy rate is comparable to that reported by Jones et a. for phases I and II of 98 (6%), the Norfolk group achieved a substantially higher pregnancy/transfer rate (7 of, or %, versus 9 of 5, or 7%). These two systems differ in many technical aspects (distance of the laboratory from the operating room, composition of media, and incubation conditions) in addition to the quantity of hmg used for ovulation induction as well as the time lag between the last ampule of hmg and the injection of hcg. Therefore, it is impossible to isolate one variable as the main contributing factor to the discrepancy in pregnancy/transfer rates between the two IVF centers; however, the fact that our pregnancy rate was lower than expected in view of the high propor-' tion of women transferred with multiple embryos may be attributed to two factors: the quality of oocytes and embryos and the effect of the high' dose of hmg administered on the endometrium. Vol. 40, No.6, December 98 Because no conceptions occurred when one or two embryos were transferred, it may be implied that high embryonic loss occurred in this system. 4 The quality of the transferred oocytes can Figure Cleaved oocytes of a successful pregnancy (case 9). (A), Low magnification (x 50) of cleaved oocytes. (B), High magnification (x 400) of the same oocytes showing well-defined cleavage planes of 4-cell-stage embryos (Hoffman Interference Optics). Laufer et al. Human menopausal gonadotropin in IVF 79

7 best be exemplified in women with multiple-embryo transfers resulting in a successful ongoing pregnancy. In these cycles the uterine factor is assumed to be equally favorable for all transferred embryos of good quality. Recently Speirs et a. 5 described a mathematical model for estimating embryonic health and quality. This model suggests that a high proportion of 8ingleton pregnancies following multiple transfers reflects lowquality embryos and is not influenced by the uterine milieu. In the eight ongoing pregnancies that we report, 8 - to 6-cell embryos were transferred, of which only (5%) of the extra 0 embryos continued to develop up to the eighth week. Because the fertilization and cleavage rates in our system are high, it is possible that although the methods used for embryo handling and culture do not affect the early cleavages following fertilization, they may still interfere with continued later development in utero. Media composition and preparation as well as quality control are crucial to the success ofivf. A uniform medium was used for both M and GM, with only the addition of the women's own serum-lo% and 0%, respectively. 'I'his, and the deletion of the paraffin oil coverage of the medium, simplifies the procedure by eliminating steps in which human errors can occur. The use of highly purified water has been emphasized in IVF media preparation.,, 0 Water distilled five times in glass is currently being used in most laboratories. Glass-distilled water may, however, contain particulate and volatile organic impurities that could affect cleavage rates despite rigorous redistillation. I6 The use of ultrapure highpressure liquid chromatography grade water, which is commercially available, has standardized this component of our medium. Even though all media tested in this series supported mouse embryo growth and fostered sperm survival with a high fertilization rate, measures of quality control should not be omitted. Such testing and quality control remain an important part of an IVF program. Recently it was reported by Garcia et a. 7 that in hmg-treated women, endometrium obtained between the first and fourth days of the luteal phase showed.advanced maturation. It was hypothesized that this effect may have some benefit for embryo implantation after IVF. The use of high-dose hmg in our system is associated with high levels of E both in the preovulatory phase as well as in the luteal phase. It may be that the uterine milieu is adversely affected by these sustained high levels of E. It is concluded that the use of high-dose hmg precludes the need for serum or urine LH monitoring because the occurrence of spontaneous ovulation is low. This regimen is safe and was associated with only one case of mild ovarian hyperstimulation syndrome. It is valuable in increasing the number of aspirated oocytes and embryos and the percentage of women undergoing multiple embryo transfer, thus compensating in part for the high embryonic loss involved with IVF by multiple oocyte transfer. ADDENDUM Since the submission of this manuscript, seven normal babies (four males and three females) have been born. The most recent pregnancy is currently at weeks of gestation. REFERENCES. Trounson AO, Mohr LR, WoodC,Leeton JR: Effect of delayed insemination on in vitro fertilization,culture and transfer of human embryos. J Reprod Fertil 64:85, 98. Jones HW Jr, Jones GS, Andrews MC, Acosta A, Bundren C, Garcia J, Sandow B, Veeck L, Wilkes C, Witmyer J, Wortham JE, Wright G: The program for in vitro fertilization at Norfolk. Fertil Steril 8:4, 98. Edwards RG, Purdy JM: Human Conception In Vitro. London, Academic Press, 98, p 9 4. Garcia J, Jones GS, Wentz AC: The use of clomiphene citrate. Fertil Steril 8:707, Hammerstein J: Mode of action of clomiphene. Acta Endocrinol (Copenh) 60:65, Laufer N, Reich R, Braw R, Schenker J, Tsafriri A: Effect of clomiphene citrate on preovulatory rat follicles in culture. BioI Reprod 7:46, Laufer N, Pratt B, DeCherney AH, Merino M, Naftolin F, Markert CL: The in vivo and in vitro effect of clomiphene citrate on ovulation and the development of cultured fer tilized oocytes. Am J Obstet Gynecol. In press 8. Laufer N, ShapiroE, SchenkerJG: A computerized system for the evaluation of HMG ovulation induction. Presented at the Tenth World Congress on Fertility and Ste rility, August 7 to 4, 980, Madrid, Spain. Abstract 9. Laufer N, Haseltine FP, DeCherney A: The value of serum 7J-estradiol in predicting follicular maturation in cycles induced by hmgihcg treatment is limited. Fertil Steril (Abstr) 9:45, Lopata A, Johnston IWH, Hoult IJ, Speirs AL: Pregnancy following intrauterine implantation of an embryo obtained by in vitro fertilization of a preovulatory egg. Fertil Steril : 7, 980. Marrs RP: Clinical application of techniques used in human in vitro fertilization research. Contemp Obstet Gynecol 0:9, Laufer et ai. Human menopausal gonadotropin in IVF Fertility and Sterility

8 . Steptoe PC, Edwards RG: Birth after the reimplantation of a human embryo. Lancet :66, 978. Steptoe PC, Edwards RG: Laparoscopic recovery of preovulatory human oocytes after priming of ovaries with gonadotropins. Lancet :68, Biggers JD: In vitro fertilization and embryo transfer in human beings. N Engl J Med04:6, Speirs AL, Lopata A, Gronow MJ, Kellow GN, Johnston WIH: Analysis of the benefits and risks of multiple embryo transfer. Fertil Steril 9:468, Runser DJ: Maintaining and Trouble-Shooting HPLC Systems. New York, Wiley & Sons, 98, p 7. Garcia JE, Acosta AA, Jones HW Jr: Advanced endometrial maturation after ovulation induction with hmg/ hcg for in vitro fertilization. Fertil Steril (Abstr) 9:46, 98 Vol. 40, No.6, December 98 Laufer et al. Human menopausal gonadotropin in IVF 74