Proc. Nati. Acad. Sci. USAVol. 81, pp. 2218-2222, April 1984Medical Sciences

Birth of rhesus monkey infant after in vitro fertilization andnonsurgical embryo transfer

(oocytes/gonadotropin/ spermatozoa/caffeine/culture)

BARRY D. BAVISTER, DOROTHY E. BOATMAN, KEVIN COLLINS, DONALD J. DIERSCHKE, ANDSTEVEN G. EISELEWisconsin Regional Primate Research Center, University of Wisconsin-Madison, Madison, WI 53715-1299

Communicated by Robert P. Hanson, December 22, 1983

ABSTRACT The birth of a rhesus monkey resulting fromin vitro fertilization is reported. Oocytes recovered at laparos-copy from five gonadotropin-stimulated donors were insemi-nated in vitro with sperm preincubated with caffeine and dibu-tyryl cyclic AMP. After insemination, oocytes were culturedfor 33-46 hr. Twenty-two embryos were transferred nonsurgi-cally into 11 recipient females. One recipient showed signs ofimplantation but did not carry to term. A second female be-came pregnant after receiving one 4-cell and one 6-cell embryofertilized in vitro. The subsequent course of early pregnancyand embryonic and fetal development were characteristic ofnormal singleton pregnancies. A healthy term male infant wasdelivered by Cesarean section 176 days after fertilization. Thisbirth has validated our procedures for in vitro fertilization ofrhesus monkey gametes and provides an experimental modelfor studies of early embryonic development in primates.

There is a long-standing need for models using nonhumanprimates to supply basic information on fertilization and ear-ly embryogenesis. This need has been increased by the clini-cal applications of in vitro fertilization (IVF) and embryotransfer (ET) for the treatment of human infertility. For ex-ample, improved pregnancy rates in humans might be at-tained by using data from experiments on prolonged embryoculture, embryo freeze storage, or fertilization and earlycleavage development in a host (xenogenous) species (1).However, serious ethical and legal questions are raised bypotential experimentation on human preimplantation embry-os. Use of nonhuman primates obviates these societal reser-vations and permits the design of controlled experiments.Secondarily, the development of IVF and ET procedures innonhuman primates could provide a tool to create largepools of genetically valuable animals for use in biomedicalresearch. Additionally, IVF of oocytes followed by transferof embryos to surrogate mothers of a closely related speciescould offer a means to preserve primates that are threatenedby extinction.As a first step towards detailed analytical studies of pri-

mate fertilization and preimplantation embryogenesis, pro-cedures must be devised for achieving these events routinelyin vitro. IVF has been reported for oocytes from squirrelmonkeys (2), baboons (3), chimpanzees (4), cynomolgusmonkeys (5), and rhesus monkeys (6). However, there areonly two reports of IVF monkey embryos undergoing regu-lar cleavage in vitro-i.e., to the 8-cell stage or beyond (5, 6).To date, there has been but a single report of a nonhumanprimate (baboon) born after transfer of IVF embryos (3).We have devised procedures for IVF and embryo culture

using the rhesus monkey (6). In this species, the average du-ration of the menstrual cycle (28 days), the regulation of folli-culogenesis, and the control of ovulation are all very similar

to these processes in women (7). Because the rhesus hasserved as a model for early human development, extensivedata on postnatal behavior and development are available(8). Thus, even subtle developmental anomalies could bereadily detected if they were to occur in offspring resultingfrom IVF and ET. We have previously reported normalcleavage development of IVF rhesus oocytes up to the moru-la stage (6). However, conclusive evidence for normal fertil-ization and embryogenesis can be obtained only from birthof normal offspring after ET. We now report the birth of ahealthy rhesus male infant after nonsurgical (intrauterine)transfer of two IVF embryos to a recipient female. This birthprovides validation of the IVF procedures used in our labo-ratory and offers an experimental model for studies on fertil-ization and embryonic development in primates.

MATERIALS AND METHODS

All female monkeys used in this study were housed separate-ly from males. Procedures for recovery of oocytes from go-nadotropin-stimulated rhesus monkeys, preparation of sper-matozoa, IVF, and embryo culture were all as previouslydescribed (6). Five female rhesus monkeys were each givena total of 2150 international units of pregnant mare serumgonadotropin (PMSG) from day 4 of the menstrual cycle (day1 = first day of menses) through day 15. On day 16, 4000international units of human chorionic gonadotropin was giv-en, and aspiration of follicles during laparoscopy was per-formed 30 hr later. Oocytes were incubated individually indrops of culture medium TALP (9). After 5-7 hr, oocytedrops (90 Al) were inseminated with 10 1.l of washed ejacu-lated spermatozoa that had been incubated with caffeine anddibutyryl cyclic AMP (1 mM each). After 20-hr incubation ofoocytes with sperm, oocytes were transferred to fresh dropsof TALP containing 20% heated (560C for 30 min) rhesusblood serum. Oocytes and embryos were examined at inter-vals with a Nikon inverted microscope equipped with phase-contrast and Nomarski optics and a temperature-controlled(370C) environmental chamber.Eleven surrogate recipients for ET were chosen by timing

of the sex-skin breakdown (10). Recipient females wereanesthetized with ketamine (Vetalar, Parke-Davis) duringthe transfer procedure. Nine recipients received two embry-os each, one received three embryos, and one received asingle embryo. For the transfer procedure, embryos wereheld in Hepes-buffered TALP (6) containing 5% heated rhe-sus blood serum. An 18-gauge blunt-end steel hypodermicneedle was carefully guided through the tortuous cervix. Astretched-out polyethylene catheter (Intramedic PE-50) con-taining the embryos in a drop approximately 1.5 cm from thetip was then passed through the steel cannula until the tip lay

Abbreviations: IVF, in vitro fertilization; ET, embryo transfer;PMSG, pregnant mare serum gonadotropin; MCG, monkey chorion-ic gonadotropin; PT, post-transfer.

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Table 1. Summary of embryo transfer experiments with IVF rhesus monkey embryos

Days betweenNo. ovulation and

recipients transfer* Outcome

4 0 One term pregnancy,t three started menses PT day 14-174 +1 or +2 Started menses PT day 10-131 +4 Started menses PT day 59t1 -10§ Started menses PT day 261 -5§ Started menses PT day 17

PT day, embryo post-transfer day.*Estimated day of ovulation was day of sex-skin breakdown (10).tLive birth, 704-g male (Petri).tTwo positive signs of pregnancy: uterine enlargement PT days 19-32 and long implantation-typebleeding. No significant increase in serum MCG by PT day 21.§Sex-skin pattern irregular; day of ovulation determined retrospectively.

in the uterine fundus. The embryos were expelled in about0.05 ml of medium, then the catheter and cannula were with-drawn simultaneously while another 0.05 ml of medium wasinjected.Blood samples were drawn from each of the recipient fe-

males at the time of ET and subsequently every other day.Serum monkey chorionic gonadotropin (MCG) assays wereperformed (11). A house standard preparation was obtainedfrom pooled rhesus pregnancy serum (days 19-23) and wasused as homologous reference (12). Data are expressed as ,.lof house standard preparation per ml of serum; the averagewithin-assay variation was 4.3%. Serum progesterone wasmeasured by using a specific radioimmunoassay (13); thewithin-assay variation was 10%. Total serum estrogens weremeasured by radioimmunoassay (14); the within-assay varia-tion was 5.4%. The blood of surrogate and genetic parentsand offspring was typed by using a simple hemagglutinationtechnique (15).

RESULTSA total of 22 embryos were transferred to 11 recipients. Tenof the embryo transfers were performed 33-46 hr after in-semination of oocytes (average 41 hr). With the exception ofone 2-cell embryo, which was transferred with two others,

all embryos were at the 4- to 8-cell stage (mean cell number5.45). Eight of the 11 females received embryos on the day ofestimated ovulation or within 2 days after ovulation (Table1). One of these transfers resulted in a term pregnancy. Theremaining three transfers were less synchronized with theestimated time of ovulation. The female that received em-bryos 4 days after the estimated time of ovulation showedtwo positive signs of pregnancy before returning to normalcycling on day 59: uterine enlargement felt by palpation onpost-transfer (PT) days 19-32 and a long "implantation-type" bleeding. There was no significant increase in serumMCG by PT day 21.The animal that maintained a pregnancy to term received

one 4-cell and one 6-cell embryo 46 hr after in vitro insemina-tion of oocytes. Fig. 1 shows these same embryos at 41 hrafter insemination. The transfer was performed on the recipi-ent's estimated day of ovulation (Table 1). The recipient'sserum steroid hormone and chorionic gonadotropin profilesfor the first 28 days PT are shown in Fig. 2. On the day oftransfer (PT = 0), serum progesterone was 4.41 ng/ml, andthe concentration had dropped to 0.69 ng/ml by the time ofthe corpus luteum rescue (16) on PT days 8-9. The peak ofestrogens associated with early pregnancy, 250 pg/ml, oc-curred on PT days 16-18. MCG reached maximal levels on

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FIG. 1. IVF rhesus embryos cotransferred to recipient. (A) Four cells and (B) six cells. One of these two produced a normal infant.Photographed 41 hr after insemination. (Optical magnification x66; final magnification x380.)

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300 7'

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FIG. 2. Serum estrogen, progesterone, and MCG concentrations during days 0-28 after ET in the female that maintained a term pregnancy.

PT days 17-19 and had dropped to near baseline values byPT day 28. Firmness of the uterus was detected by rectalpalpation on PT day 28. An ultrasound scan 40 days PTshowed the presence of a single embryo, crown-rump length19 mm, with a beating heart. A second ultrasound scan on

PT day 99 showed a normal-looking fetus with a cranial di-ameter of 3.6 cm. A male infant, "Petri," weighing 704 g,was delivered by Cesarean section 174 days PT (Fig. 3).Neurological reflexes, which included righting, grasping,rooting, and plantar flexion, were normal when tested within1 hr of birth. Results of blood typing the surrogate mother,the genetic mother and father, and Petri for 11 blood groupswere consistent with the offspring's known genetic parent-age and excluded the surrogate mother from being the genet-ic parent (Table 2).

.44

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FIG. 3. The rhesus infant, Petri, shortly after birth.

DISCUSSIONFour major points have been demonstrated by the birth ofthe infant rhesus monkey: first, normal offspring can be pro-duced from rhesus oocytes fertilized in vitro; second, oo-

cytes obtained from monkeys stimulated with exogenous go-nadotropin (PMSG) can produce normal offspring; third,normal development can take place after fertilization of oo-

cytes by spermatozoa stimulated with caffeine and dibutyrylcyclic AMP; and fourth, early cleavage stage nonhuman pri-mate embryos can be transferred nonsurgically to the uterus,where they can continue their development to the implanta-tion stage and fetuses. These main points will be discussed inrelationship to the factors that have been identified as beingcentral to the establishment of pregnancy after IVF and ET:the quality of the embryos (18, 19), the synchrony of the re-

cipients (20), and the mechanics of the transfer procedure(19, 21).There are two confirming pieces of evidence that the rhe-

sus infant, Petri, was the result of IVF. His male gender ex-

cluded parthenogenetic activation of the egg. Since the H2blood group allele in his genotype (Table 2) was carried onlyby the egg donor, the surrogate recipient was excluded as thepossible genetic mother (15, 17). Live offspring have beenborn after IVF in six other species: uncounted numbers ofmice, rats, and rabbits (22-24); more than 100 humans (25), 1calf (26), and 1 baboon (3). The strategy for IVF differs sig-nificantly for the normally monotocous species (primates

Table 2. Confirmation of parentage by blood typingSource Abbreviated blood type*

Sperm donor GI/G2 H'/H1 M3/- Rr/RrEgg donor G2/G2 H'/H2 M1/M3 Rr/RrSurrogate mother G2/G2 H'/H1 M3/- Rl/-Offspring (Petri) G'/G2 H'/H2 M3/- Rr/Rr*Loci omitted were uninformative; -, ambiguous genotype [for ex-ample, M3/- = M3/M3 or M3/Mm (17)].

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and cows) from that usually employed for the polytocouslaboratory animals. In the former, follicular oocytes are har-vested prior to ovulation, while in the latter, mature oocytes(ova) are nearly always recovered from the oviducts. Thus,the initial maturity and quality of the oocytes are different.Oocyte maturity at the time of collection may be critical forobtaining quality embryos and offspring after IVF in primatespecies (18, 19, 27). Human oocytes derived from follicleswith steroid hormone levels characteristic of mature follicleshave a higher incidence of establishing a pregnancy after fer-tilization (19). Rapidly cleaving embryos are more frequentlyderived from "mature" follicles, and establishment of preg-nancy after transfer is also correlated with rapid cleavage ofembryos (18). There are two main sources of retardation ofcleavage in vitro: factors intrinsic to the oocyte itself (suchas oocyte maturity), and defects in the culture environment(6, 19). In vivo fertilized rhesus embryos have shown retard-ed cleavage when placed into a deficient culture environ-ment (28). The maturational status of rhesus oocytes ob-tained by our procedure and their unretarded cleavage devel-opment to the 8-cell stage after IVF have been described (6).Equivalent embryos were transferred to recipients in thepresent study. At the time of transfer, 33-46 hr after in vitroinsemination, 18/22 of the embryos were in the 4- to 8-cellstage. After transfer, there was no evidence for retarded ear-ly embryonic development in the full term pregnancy. Thesteroid hormone and chorionic gonadotropin (MCG) profiles(Fig. 2) showing the rescue of the corpus luteum (16) onpregnancy days 9-10 (pregnancy day = PT day + 2 in vitroembryonic days), the MCG peak on pregnancy day 19-21,and the temporal relationships between estrogens, proges-terone, and MCG, were all as expected for normal rhesuspregnancies (12, 16). The crown-rump length of the embryoon pregnancy day 42 (19 mm) was in the expected range forin vivo fertilized embryos (29). The term-pregnant animalwas one of several recipients to receive embryos from thesame egg donor. Judged by morphological criteria (18), sev-eral of the nonpregnant recipients received embryos as goodor better than those transferred to the pregnant animal.Clearly, factors in addition to embryo quality are also impor-tant in obtaining offspring after IVF.PMSG has been used to produce multiple embryos in cat-

tle and rodents. Some attempts have been made to usePMSG for superovulation of nonhuman primates (30-32).There has been concern for the normality of oocytes or em-bryos obtained from primates treated with PMSG and otherexogenous gonadotropins. Abnormalities of the zona pellu-cida in follicular oocytes (33) as well as abnormal cleavagestage embryos have been observed (30). The good cleavagedevelopment that we have obtained for IVF embryos afterstimulation of follicular growth with PMSG and the birth of anormal infant argue that at least some of the oocytes fromtreated animals are normal. This is consistent with the find-ing that PMSG does not result in defective oocytes in rats(34).The penetration of rhesus oocytes in vitro is enhanced by

prior treatment of rhesus sperm with caffeine and dibutyrylcyclic AMP (35). Rhesus oocytes penetrated by treatedsperm undergo normal cleavage development in vitro (6).Under certain conditions, caffeine enhances the ability ofhuman sperm to penetrate hamster zona-free ova (36, 37). Atherapeutic use for caffeine to enhance human sperm pene-tration of human eggs in vitro has been suggested (36). Sincecaffeine appears to have some teratogenic activity in mam-mals and can induce chromosomal abnormalities in cells inculture (38), its potential benefits must be weighed againstthe risk of fetal abnormalities. The rhesus infant born aftersperm and egg exposure to low doses of caffeine and dibu-tyryl cyclic AMP is normal and healthy. Caffeine treatmentof human sperm may indeed prove beneficial for IVF when

both husband and wife contribute to infertility.Bovine ET data indicate that the stage of the embryo and

the estrous cycle of the recipient should be synchronized foroptimal results (20). No similar data are available for nonhu-man primates to indicate the acceptable time for ET to recip-ients. Logically, an embryo 2 days after insemination shouldbe transferred to a recipient 2 days after ovulation. Alterna-tively, replacing the embryo at a time closer to ovulationmight compensate for possible retarded embryonic develop-ment. Eight of the 11 recipients appeared to meet either ofthese timing criteria (Table 1). Since there is no known wayto synchronize nonhuman primates artificially, the accuratedetermination of synchrony is crucial for ET to surrogates.Currently, sex-skin breakdown (10) is the most rapid meansof selecting potential recipients from the colony. While sex-skin color during the menstrual cycle has proven to be usefulin breeding rhesus monkeys (39), sex-skin breakdown is nota precise means of determining the time of ovulation on anindividual basis. Retrospective analysis showed that two re-cipients were apparently not at the predicted stage of thecycle on the day of transfer (Table 1). The animal that car-ried the term pregnancy was a model candidate for ET. Her28-day menstrual cycles were very regular and the day ofsex-skin breakdown was well demarcated. Transfer wasdone on the estimated day of ovulation (Table 1). The serumprogesterone level of 4.41 ng/ml on the day of transfer (Fig.2) was consistent with an ovulatory cycle (40). Another fe-male received embryos 4 days after the estimated day ofovulation and showed two signs of early pregnancy beforereinitiating menstruation (Table 1). While this may indicatethat some leeway is permissible in synchrony of the embryoand recipient, interpretation is difficult because pregnancydid not develop.ET using in vivo fertilized embryos within and between

nonhuman primates was demonstrated to be feasible prior tocomparable success with IVF. Surgical procedures to ex-pose uteri and oviducts were used for these transfers. In1976, in vivo fertilized morula or blastocyst stage baboonembryos were surgically transferred to the uteri of 10 surro-gate recipients. This resulted in two pregnancies and one livebirth (41). However, autotransfers of in vivo fertilized rhesusembryos to the contralateral oviducts within 30 min of recov-ery resulted in a higher pregnancy rate: 8/10 transfers result-ed in pregnancy, with 4 live births (42). A term pregnancyoccurred after a cleavage stage embryo was flushed from theoviduct of a rhesus monkey, cultured for 24 hr, then returnedto the oviduct (5). The baboon born after IVF and ET result-ed from surgical transfer of pronucleate and 2-cell-stage em-bryos to the oviduct of a surrogate (3). Surgical transfers incattle have generally been more successful than have non-surgical (cervical) procedures (21). Despite this observation,nonsurgical uterine transfers are used for human infertilitypatients (19). Abdominal surgery can cause adhesions lead-ing to infertility in women. Abdominal postsurgical adhe-sions can also occur in monkeys. Nonsurgical transfer pro-cedures would permit safer use of prime breeding animals forsurrogates. However, the tortuous anatomy of the rhesuscervix (and that of some other macaques) (43) is an impedi-ment for cervical access to the uterus. Nonetheless, we haveobtained one early pregnancy and one live birth in 11 nonsur-gical transfers of IVF embryos to surrogates (Table 1), a ratecomparable to that after surgical transfer of in vivo fertilizedbaboon conceptuses to uteri of surrogates (41). Undoubted-ly, improvements in transfer techniques will be needed tooptimize pregnancy rates.Knowledge gained in the study of nonhuman primate IVF

and ET concerning folliculogenesis, early fertilization events,embryogenesis, and implantation would be expected to haveapplications to human reproduction and fertility. Beyond that,a more abundant supply of early nonhuman primate embryos

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for in vitro investigations would make it feasible to study theetiology of birth defects in primates, to examine primategene regulation and differentiation in nontransformed cells,to produce identical twins and age-matched full siblings forresearch, to produce groups of primates with specific genetictraits, and to diversify breeding stocks of primates in zoosand research institutions through the shipping of stored em-bryos.

We are grateful to Dr. W. H. Stone and C. Blystad for blood typ-ing analysis; to Dr. W. D. Houser for veterinary services; to Dr.J. A. Robinson, Dr. W. E. Bridson, G. Scheffler, and F. Wegner forhormone assays; to R. Pierson for ultrasonography; to M. Hempel,R. Dodsworth, and R. Pape for technical services; and to C. War-field for secretarial assistance. This work was supported by the Wis-consin Regional Primate Research Center (grant RR-00167 from theAnimal Resources Branch of the National Institutes of Health) andgrant HD-14765 from the National Institutes of Health (to B.D.B.).This is Wisconsin Regional Primate Research Center publication no.23-018.

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