Successful in vitro fertilization and embryo transfer in cynomolgus monkeys

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1 FERTILITY AND STERILITY Copyright e 984 The American Fertility Society Vol. 4, No.5, November 984 Printed in U.S.A. Successful in vitro fertilization and embryo transfer in cynomolgus monkeys Jose P. Balmaceda, M.D.*t Thomas B. Pool, Ph.D.:j: Jaime B. Arana, M.D. * Teri S. Heitman, B.S.:j: Ricardo H. Asch, M.D.* The University of Texas Health Science Center at San Antonio, San Antonio, Texas We have started an in vitro fertilization program in cynomolgus monkeys in an effort to develop an appropriate animal model to improve our knowledge of early embryonic development. In 6 of 5 animals treated with menopausal gonadotropins, preovulatory follicles developed. Follicular aspiration was performed at laparotomy after human chorionic gonadotropin injection. A total of 99 follicles were aspirated, and 5 oocytes were recovered. Oocytes were cultured in ml of growth medium or 00 ILL droplets of medium under mineral oil. Semen samples were obtained by electroejaculation, and the oocytes were inseminated 4 to 4 hours after aspiration. Culture under mineral oil significantly increased the fertilization and cleavage rates. Of 68 embryos produced, 4 have been used in 0 embryo transfers, resulting in two pregnancies. Fertil SteriI4:79, 984 Since the report of the first successful pregnancy obtained in humans with in vitro fertilization (lvf) and embryo transfer (ET) by Steptoe and Edwards, l these techniques have become an important tool in the treatment of infertile couples. However, the obvious ethical and medical limitations regarding the experimental use ofhuman embryos represent important obstacles in the improvement of IVF techniques for the clinical setting. It is crucial, then, to develop appropriate animal models for IVF through which our Received May 5, 984; revised and accepted July 5, 984. *Department of Obstetrics and Gynecology, Division ofhuman Reproduction. treprint requests: Jose P. Balmaceda, M.D., Department of Obstetrics and Gynecology, The University of Texas Health Science Center at San Antonio, 770 Floyd Curl Drive, San Antonio, Texas :j:department of Cellular and Structural Biology. Vol. 4, No.5, November 984 knowledge of fertilization and embryonic development can be increased. Ideally, such models will also allow investigators to experiment with different methods of ovulation induction, embryo culture, and ET that can have immediate clinical applications. A number of investigators have studied IVF and ET in nonhuman primates. Successful IVF of nonhuman primate oocytes in the squirrel monkey,, baboon,4 cynomolgus monkey,5 rhesus monkey, 6 and chimpanzee 7 has been attained. More recently, the birth of a baboon and of a rhesus monkey conceived after IVF and ET have beeri reported in the literature.s, 9 We have chosen to work with the cynomolgus monkey, Macaca fascicularis, because of the similarities of their reproductive cycle with the human cycle and because of their availability. We have accumulated experience in the laparoscopic examination and dating of ovulation in this species that BaImaceda et ai. NF and ET in cynomolgus monkeys 79

2 has greatly facilitated the development of ovulation induction and egg recovery protocols for IVF. MATERIALS AND METHODS Sexually mature female cynomolgus monkeys, weighing to 6 kg and experiencing regular menstrual cycles, were used in this study. They were caged individually under controlled conditions of ± C and 50% relative humidity and were exposed to a 4-hour light/0-hour dark photoperiod. Commercially prepared pelleted monkey chow was supplemented twice weekly with fresh fruit and chewable multivitamins with iron and minerals. Tap water was available ad libitum. Daily vaginal swabbings were used to detect the onset and duration of menses. INDUCTION OF OVULATION In a preliminary series of experiments, we compared the administration of clomiphene citrate versus human menopausal gonadotropin (hmg) (Pergonal, Serono Laboratories, Inc., Randolph, MA) to induce superovulation in the monkeys. Induction of ovulation with hmg consistently yielded a larger number of mature oocytes at aspiration; for this reason, it was selected as the drug for our protocol. The animals received one half of an ampule of hmg (7.5 IU luteinizing hormone; 7.5 IU follicle-stimulating hormone) daily from days through 8 of the cycle. A laparoscopy, to determine follicular development, was performed on day 9 of the cycle. If follicular development was considered to be satisfactory, human chorionic gonadotropin (000 IU, intramuscularly) was given at 8:00 to 0:00 P.M. of the same day of the cycle. FOLLICULAR ASPIRATION Laparotomy and follicular aspiration were performed 4 to 8 hours after the injection of human chorionic gonadotropin. During the procedure, the animals were maintained under ketamine hydrochloride anesthesia (0 to 5 mglkg, intramuscularly). After the abdominal wall was opened, the genital tract was isolated by packing the bowel with wet sponges. The ovaries were then suspended from the utero-ovarian ligaments, and individual follicles were aspirated with a 0-gauge needle (45-degree bevel) attached to a -ml syringe containing 0. ml of aspi- 79 Balmaceda et al. NF and ET in cynomolgus monkeys ration medium (described below). The syringes were transferred immediately to a sterile hood, and the contents were emptied into plastic dishes. CULTURE AND ASPIRATION MEDIA The medium used for oocyte culture, embryo culture, and ET was a modified Tyrode's solution (TALP) developed by Bavister and Yanagimachi. lo The medium used for follicular aspiration and sperm preparation was TALP-HEPES. TALP was prepared fresh weekly, with the exception of labile components (bovine serum albumin, sodium pyruvate), which were added immediately prior to use. Complete TALP was adjusted to 80 mosm, ph 7.4, and was supplemented with % heat-inactivated serum collected from midcycle females. Gentamicin sulfate was added to a final concentration of 50,...g/ml. The medium was further supplemented with glutamine (46,...g/ml). Follicular aspirates were examined in 60-mm plastic Petri dishes with a dissecting microscope for the presence of oocytes. Aspirated fluid was diluted further with 5 ml oftalp-hepes if the number of granulosa cells or erythrocytes was sufficient to preclude the rapid recovery of oocytes from the follicular fluid. Upon identification, 00- cytes and associated cumulus cells were transferred immediately to dishes containing either ml of complete growth medium (TALP plus all supplements) alone or to dishes containing 00-,... droplets of «omplete growth medium under a -ml mineral oil overlay (paraffin oil, laboratory grade, lot #7550; Fisher Scientific, Fair Lawn, NJ). This batch of oil was determined to be nontoxic in preliminary experiments and is used without pretreatment. All dishes were preincubated for hour at 7 C in a humidified atmosphere of 5% CO :95% air prior to use. Oocytes recovered from the aspirated follicular fluid were classified as type I, II, or III, following the criteria utilized by Bavister and co-workers 6 in the rhesus monkey (Fig. ). SPERM PREPARATION AND INSEMINATION IN VITRO Semen from male monkeys of known fertility was collected by electroejaculation into ml of TALP-HEPES in sterile plastic tubes. The freshly collected semen was then suspended in 0 ml of TALP-HEPES prior to centrifugation at 00 x g for 5 minutes. The supernatant was discarded, and the pellet was resuspended in to ml of fresh TALP-HEPES. Sperm concentrations were Fertility and Sterility

3 Table. Fertilization and Cleavage According to Culture Conditions Culture conditions Growth medium ( ml) Growth medium (00 f.l) under mineral oil No. of oocytes Inseminated Fertilized Cleaved 9 (9%) 6 (5%) 7 (57%) 6 (48%) Total 5 96 (8%) 68 (7%) evidence of fertilization, during the first 4 hours following insemination were reinseminated with sperm from a fresh sample for an additional hours. EMBRYO TRANSFER ET was performed via a laparotomy procedure during which the embryos were placed in the fallopian tube. Embryos were picked from the culture dishes using a micropipette (Quick-SGT Micro/Pettor model 800A; Scientific Manufacturing Industries, Emeryville, CA) in f-li of culture medium. The plastic tip of the micropipette was introduced through the fimbriated end of the tube, and the embryos were delivered in the midampullary portion. Figure (a), Type I oocyte surrounded by a corona and loose cumulus. (b), Type II oocyte surrounded only by a corona. (e), Type III oocyte. determined with a hemocytometer, and the suspension was further diluted with TALP-HEPES to yield 0 7 sperm/ml. The diluted suspension was returned to 7 C and incubated for to 4 hours, then incubated an additional to.5 hours in TALP-HEPES containing mm caffeine and mm dibutyryl cyclic adenosine monophosphate (dbcamp) as described for the rhesus monkey.6 Aliquots of sperm, incubated in the presence of caffeine and dbcamp, were added to oocyte cultures between 4 and 8 hours after follicular aspiration. The volume of the aliquot of sperm suspension was 00 f-li or less so as to dilute the caffeine and dbcamp by a factor of 0 or more.6 Oocytes were incubated at 7 C in 5% CO :95% air in the presence of sperm for to 4 hours and were then transferred to fresh growth medium. In some cases, eggs that showed evidence of maturation (extrusion of the first polar body), but not RESULTS Twenty-five monkeys have undergone ovarian stimulation with hmg up to now in our program. Nine were considered not to have preovulatory follicles at the time of laparoscopy and were dropped from the study. Sixteen (64%) animals underwent laparotomy for follicular aspiration. A total of 99 follicles were aspirated, and cytes were recovered. Classification of the oocytes at the time of recovery was as follows: 7 type I, 5 type II, and 5 type III. AU classified oocytes recovered were inseminated. Ninety-six of the 5 oocytes fertilized, and 68 cleaved at least Table. Fertilization and Cleavage Rates According to Oocyte Class No. of oocytes Oocyte class Inseminated Fertilized Cleaved I 9 (7%) 59 (64%) 5 (55%) II 4 (9%) 7 (9%) 6 (5%) III (0%) 7 (54%) 5 (8%) Total Vol. 4, No.5, November 984 Balmaeeda et ai. IVF and ET in cynomolgus monkeys 79

4 Table. Results of Embryo Tronsfers Animal no. 4 a 5 6 7a,b 8 ga,b loa,b No. of embryos transferred 4 4 asynchronized recipient, brhesus monkey (Macaca mulatta). Pregnancy Yes Yes once. As shown in Table, culture conditions importantly influenced the rate of fertilization and cleavage, The percentage of oocytes that fertilized and underwent cellular division was significantly higher in the group cultured under mineral oil (P < 0.00). Table presents the fertilization and cleavage rates of oocytes cultured under oil, categorized by type. Although fertilization and cleavage of type loocytes were higher than for types II and III, the difference was not statistically significant. The number of embryos obtained from individual animals has ranged from to 0. A total often ETs have been performed in the program up to now (Table ). All embryos transferred were at the - or 4-cell stage (Fig. ). Four transfers were done with synchronized recipients, three of them rhesus monkeys. As shown in Table, two pregnancies have occurred. The pregnancy of animal resulted in a spontaneous abortion, after a gestational sac had been documented by serial ultrasound examination. Animal 7 (a rhesus recipient) was delivered at term (56 days of gestation) by cesarean section. citrate. In addition, the use of gonadotropins needed less cumbersome monitoring to determine the time of follicular aspiration. Previous publications have suggested that spontaneous ovulation, following an endogenous luteinizing hormone peak, is rare in monkeys in which several preovulatory follicles have been induced with the use of gonadotropins. ll Our' results appear to support this observation, because none of the 6 animals aspirated showed signs of ovulation at the time of laparotomy. The number'of oocytes recovered ranged from 4 to 6 per animal, with a mean of 8.7, suggesting. that hyperstimulation was the rule. However, the proportion of mature type I oocytes (7%) was similar to that reported in human programs,, and postmature or degenerating oocytes were a rare occurrence. Moreover, the rates of fertilization and cleavage of oocytes cultured under oil were in the same range as those published by DISCUSSION In the present study, we describe the method utilized for successful IVF and ET in cynomolgus monkeys. Although Kreitmann et al. 5 reported a pregnancy following the transfer of an oocyte fertilized in vivo in this species, to our knowledge, ours is the first report of a pregnancy occurring after transferring embryos produced in vitro. As we stated above, we chose to use hmgs to stimulate follicular development because their use consistently induced a larger number of follicles in this species, as compared with clomiphene 794 Balmaceda et ai. IVF and ET in cynomolgus monkeys Figure (a), A mature oocyte. (b), A -cell embryo. (c), A 4-cell embryo. (d), An 8-cell embryo. Transfers were all performed using - or 4-cell embryos. Fertility and Sterility

5 presenting a germinal vesicle (a and b) and, in another case, a polar body (c and d), suggesting a significant difference in maturity. The experimental model described in this report may prove valuable in familiarizing ourselves with techniques such as cryopreservation and the micromanipulation of primate embryos. REFERENCES Figure Photomicrographs of type III oocytes with a germinal vesicle (a and b) and a polar body (c and d). Bavister et a. 6 in the rhesus monkey. The main advantage of using mineral oil overlay is that the ph of the medium is stabilized, because the rate of gas exchange of the medium with the environment is slowed. TALP, once equilibrated with CO, maintains a constant ph much longer under an oil overlay when removed from the incubator than does TALP without an overlay. In our studies, the incidence of cleavage fell.markedly if the ph of the culture medium was not maintained within strict.limits. The use of the classification of oocytes proved to be of value when applied in our system. In general, type I oocytes or those surrounded by a corona radiata and loose cumulus were the most mature, and their rates of fertilization and cleavage were higher. The relation of the type of oocyte with maturity, though, was less direct in type II and III oocytes. Figure shows photomicrographs of type III oocytes immediately after aspiration,. Steptoe PC, Edwards RG: Birth after reimplantation of a human embryo. Lancet :66, 978. Gould KG, Cline EM, Williams WL: Observations on the.induction of ovulation and fertilization in vitro in the squirrel monkey (8aimiri sciureus). Fertil Steril 4:60, 97. Kuehl TJ, Dukelow WR: Time relations of squirrel monkey (8aimiri sciureus) sperm capacitation and ovum maturation in an in vitro fertilization system. J Reprod Fertil 64:5, Gould KG: Fertilization in vitro of nonhuman primate ova: present status and rationale for further development of the technique. In Report of the Ethics Advisory Board, Appendix: HEW Support of Research Involving Human in Vitro Fertilization and Embryo Transfer. Washington, D.C., U.S. Government Printing Office, No. 4, 979, P 5. Kreitmann 0, Lynch A, Nixon WE, Hodgen GD: Ovum collection, induced luteal dysfunction, in vitro fertilization, embryo development and low tubal ovum transfer in primates. In In Vitro Fertilization and Embryo Transfer, Edited by ESE Hafez, K Semm. Lancaster, MTP Press Ltd., 98, p 0 6. Bavister BD, Boatman DE, Leibfried L, Loose M, Vemon MW: Fertilization and cleavage of rhesus monkey oacytes in vitro. BioI Reprod 8:98, Gould KG: Ovum recovery and in vitro fertilization in the chimpanzee. Fertil Steril 40:78, Clayton 0, Kuehl TJ: The first successful in vitro fertilization and embryo transfer in a nonhuman primate. Theriogenology :8, Bavister BD, Boatman DE, Collins K, Dienske DJ, Eisler IS: Birth of Rhesus monkey infant afterivf and non-surgical embryo transfer. Proc Nat! Acad Sci USA 8:8, Bavister BD, Yanagimachi R: The effects of sperm extracts and energy sources on the motility and acrosome reaction of hamster spermatozoa in vitro. Bioi Reprod 8:8, 977. Schenken RS, Hodgen GD: Follicle-stimulating hormoneinduced ovarian hyperstimulation in monkeys: blockade of the luteinizing hormone surge. J Clin Endocrinol Metab 57:50, 98. 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 oocytes in a program of in vitro fertilization. Fertil Steril 9:594, 98. Laufer N, DeCherney 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 40:74, 98 Vol. 4, No.5, November 984 BaImaceda et ai. IVF and ET in cynomolgus monkeys 795