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Reproductive Technology |
Division of Reproductive Sciences3
Division of Animal Resources,4 Oregon National Primate Research Center, Beaverton, Oregon 97006
Departments of Obstetrics/Gynecology and Physiology and Pharmacology,5 Oregon Health and Science University, Portland, Oregon 97201
New England Clinic of Reproductive Medicine, Inc.,6, Reading, Massachusetts 01867
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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assisted reproductive technology, embryo, in vitro fertilization, pregnancy
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Development of the ARTs in humans for the treatment of infertility has resulted in dramatic progress over the past 20 yr both in pregnancy establishment and in the types of infertility that can be treated. Rapid advances have occurred in procedures for ovarian follicular stimulation; treatment of male infertility by intracytoplasmic sperm injection (ICSI) with ejaculated, epididymal, or testicular sperm both fresh and frozen; culture-medium development for the production of blastocysts; low-temperature storage of sperm and embryos; and nonsurgical embryo transfer at the blastocyst stage [3]. Unlike in many fields where knowledge flows from NHP research to clinical application in humans, the development of the ARTs in NHPs historically postdates human application. Moreover, the experience with the ARTs in NHP is much more limited than that in humans. Nevertheless, several significant accomplishments have been achieved and various methods to produce pregnancies have been described [4–6]. While a few reports in New World monkeys and Great Apes exist, most research efforts have focused on Old World macaques [4], with the rhesus monkey the primary subject and the focus of the present report.
The first successful birth of a rhesus macaque following in vitro fertilization (IVF) was reported in 1984 by Bavister and coworkers [7]. However, the total number of infants born to date probably does not exceed 100. Development of the ARTs in NHPs has been slowed by resource availability, high expense, and lack of compelling incentives to produce large numbers of monkeys using this approach. The latter limitation has recently been obviated by the need for Indian-origin, rhesus macaques carrying the class 1 MHC allele, A*01, for HIV-vaccine development research.
An accurate knowledge of pregnancy outcome is prerequisite to evaluating the impact of the ARTs on animal availability. In the human, extensive databases now exist on pregnancy outcome [3]. In general, safety and efficacy issues have largely been satisfactory, although low birth weights in singleton pregnancies have been reported along with concerns over genetic problems following the use of ICSI [8–10]. Additionally, in nonprimate animals, a large-offspring syndrome [11] has been associated with in vitro manipulation of embryos, and while this does not appear to be a problem in ART-produced children, its possible existence in NHPs has not been evaluated. Indeed, to date, there have been no published assessments of pregnancy outcomes for ART-produced monkeys involving more than a few pregnancies. Here we summarize experience at Oregon National Primate Research Center (ONPRC) occurring predominately between 1999 and 2003 with the conclusions that embryos can be produced in high efficiency using either fresh or cryopreserved sperm, that early embryonic development is adequate, although not optimized, and that 30% pregnancy rates can be achieved with either fresh or cryopreserved embryos. Moreover, pregnancy outcomes following the transfer of ART-produced embryos are similar to those observed in naturally mated, caged populations.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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All animal usage involved in these studies was reviewed and approved by our local Institutional Animal Care and Use Committee.
Controlled ovarian stimulation and oocyte recovery have been described previously [4, 12]. Briefly, cycling females were subjected to follicular stimulation using twice-daily intramuscular injections of recombinant human FSH as well as concurrent treatment with Antide, a GnRH antagonist, for 8–9 days. Females received recombinant human LH on Days 7–9 and recombinant hCG on Day 10. Cumulus-oocyte complexes were collected from anesthetized animals by laparoscopic follicular aspiration (27–29 h post-hCG) and placed in Hepes-buffered TALP (modified Tyrode solution with albumin, lactate, and pyruvate) [7] containing 0.3% bovine serum albumin (TH3) at 37°C. Tubes containing follicular aspirates were placed in a portable incubator (Minitube, Verona, WI) at 37°C for transport to the laboratory. Aspirates were sifted through a cell strainer (Falcon, 70-µm mesh size; Becton-Dickinson, Franklin Lakes, NJ). Hyaluronidase (0.5 mg/ml; Sigma Chemical Co., St. Louis, MO; in TH3) was added directly to the tubes containing aspirates followed by incubation at 37°C (30 sec) before the contents were gently agitated with a serological pipette to disaggregate cumulus and granulosa cell masses and then were poured on the strainer. Oocytes were retained in the mesh, while blood, cumulus, and granulosa cells were sifted through the filter. The strainer was immediately backwashed with TH3 and the medium containing oocytes was collected. Residual cumulus cells were removed with a small-bore pipette (approximately 125 µm in inner diameter) before recovered oocytes were examined for determination of developmental stage (germinal vesicle [GV], metaphase I [MI], or metaphase II [MII]) and quality (granularity, shape, and color of the cytoplasm). Oocytes were placed in chemically defined, protein-free HECM-9 medium [13–15] at 37°C in 5% CO2, 5% O2, and 90% N2 covered with paraffin oil (Ovoil, Zander IVF, Vero Beach, FL).
Semen was collected by penile electroejaculation [16] and allowed to liquefy at 27–32°C for 10–15 min. The liquid portion was harvested from the coagulum into a 15-ml conical centrifuge tube (Fisher Scientific, Tustin, CA) and washed twice by centrifugation at 130–150 x g for 5 min and resuspension in 5 ml TH3. Motility and concentration were evaluated microscopically and only samples with an initial motility in excess of 70% with a strong forward progression were used. For sperm cryopreservation, the washed sperm pellet was resuspended in 0.25 ml of TES-TRIS buffer containing 3% glycerol, 30% egg yolk, 20% skim milk, 0.06 M glucose and equilibrated at 4°C for 1 h [17]. This sperm suspension was frozen in 20- to 50-µl drops by placing individual drops for 10 min in small pits carved in the surface of dry ice. The goal was to create drops of 1–2 million washed sperm each, ideally in a 20-µl volume. Frozen drops (up to 10) were then transferred with precooled forceps to precooled 2-ml cryovials (Catalog # 5000-0020; Nalge Nunc International Co., Naperville, IL) and the vials were placed on a cane before being plunged into liquid nitrogen for storage. For thawing, a single pellet, retrieved from liquid nitrogen, was placed in a dry test tube, suspended in a 37°C water bath for 40 sec, and then washed in 5 ml of TH3 as described above. Individual sperm, either fresh or after cryostorage, were selected for ICSI on the basis of normal morphology and progressive motility.
ICSI, embryo cryostorage, twinning, and nuclear transfer procedures were conducted as described [4, 18]. In the majority of embryo thaw and transfer cases, embryos were thawed and placed in culture the afternoon preceding the scheduled day of transfer. Criteria to judge embryo survival the following morning were based on the following: presence of a zona pellucida, recovery of >50% of the initial number of blastomeres, and the maintenance of overall embryo quality. If no embryos survived, a second thaw with immediate transfer was undertaken, time permitting.
Embryo culture employing modified Connaught Medical Research Laboratories (CMRL; Invitrogen, Carlsbad, CA) medium on buffalo rat liver (BRL) cocultures followed previously described protocols [19]. The current approach employing HECM-9 was based on published experience [13–15]. After ICSI, injected oocytes were placed in 4-well (1 cm diameter) dishes (catalog # 176740; Nalge Nunc International) containing protein-free HECM-9 medium (0.7 ml) and covered with paraffin oil (0.3 ml; Ovoil). For extended culture, embryos at the eight-cell stage were transferred to fresh plates of HECM-9 medium supplemented with 5% fetal bovine serum and cultured for a maximum of 7 days, with medium change every other day. Embryo growth rates were based on daily assessments.
Embryo transfer was undertaken in recipients chosen on the basis of general health and physical condition, usually a record of previous pregnancy and live birth, and a history of normal ovarian cycles. Beginning 8 days after menses detection during a spontaneous menstrual cycle, blood samples were collected daily from the saphenous vein for determination of estradiol by radioimmunoassay. The LH surge was estimated to occur before the precipitous decline in serum estradiol, typically to levels below 100 pg/ml. The day when serum estradiol peaked was considered the day before ovulation (Day 1). This peak occurred on average 11 days postmenses, with a range from 8 to 17 days. Two to 6 days after the estradiol peak, fresh or frozen-thawed embryos, typically two per recipient, were transferred surgically to the oviduct ipsilateral to the ovary bearing the ovulatory stigma in anesthetized recipients as described previously [4]. A nonsurgical, transcervical approach to embryo transfer [7, 20] has also been used successfully, as described by Nusser and coworkers [19] Our laparoscopic approach to oviductal ET is described in detail. Monkeys were anesthetized with isoflurane gas vaporized in 100% oxygen and underwent comprehensive physiologic monitoring throughout the surgery, including electrocardiogram, peripheral oxygen saturation, and end-expired carbon dioxide. Orotracheal intubation and mechanical ventilation to maintain expired CO2 at less than 50 mm Hg was mandatory. After sterile skin preparation and draping, the abdomen was insufflated with CO2 at 15 mm Hg pressure and the viewing telescope was inserted via a small supraumbilical incision, with accessory ports placed in the paralumbar region. The monkey was placed in the Trendeleburg position, allowing the viscera to migrate in a cephalad direction, exposing the reproductive organs. After insertion of the telescope, the ovaries were examined with a self-retaining microretractor inserted at a high paramedian position. The transfer was conducted into the oviduct with an ovulation site on the associated ovary. The fimbria was grasped with a Patton retractor (Cook, Ob/Gyn, Spencer, IN) and placed in traction. The guide cannula was introduced into the oviduct. Typically, two ICSI or IVF embryos were transferred. To this end, embryos were removed from culture medium and transferred to a dish containing TH3 medium. The Patton polyurethane transfer catheter (Cook OB/GYN) connected to a 1-ml syringe was filled with about 0.01–0.02 ml of TH3 medium, avoiding air bubbles. Embryos were carefully loaded near the catheter tip with a total volume not exceeding 0.03 ml. The catheter was then inserted transabdominally and advanced through the fimbrium into the oviduct to a distance of 1–3 cm, where the embryos were deposited. Following transfer, the catheter was removed and carefully examined and rinsed to ensure that all embryos had been expelled. In the event of a retained embryo, a second transfer was attempted. The insufflation was reduced and the incisions were closed with interrupted absorbable suture in an intradermal pattern. Postoperative analgesia was provided through administration of buprenorphine (0.03 mg/ kg, 1 M).
Pregnancy Detection and Monitoring
To detect pregnancy, estradiol and progesterone profiles were monitored every third day after ET for 25 days, at which time the existence of a clinical pregnancy was confirmed by fetal cardiac activity as determined by ultrasonography. Confirmed pregnancies were monitored periodically throughout gestation by ultrasound. For the timed mated breeding (TMB) colony, females were palpated 30 days postpairing. All positives and questionable 30-day palpations were repalpated at 60 days. Subsequently, pregnancy was monitored by weight gain and evidence of menses or by ultrasound, if deemed clinically necessary.
Birth Weights, Gestational Age, and Growth Rates
Gestational age was measured from the middle day of pairing (pairing was usually for 3 days) for the TMB colony and from the day of ovulation for ART-related pregnancies. Computerized records of vital statistics were maintained by ONPRC. Gestation Day (GD) 165 ± 10 was considered as the normal gestational period for rhesus macaques. Delivery before 155 days was considered preterm, and before 140 days, premature [21]. Pregnancy loss in <140 days was considered a spontaneous abortion (SAB), while loss after 140 days was considered a stillborn (SB).
Statistical Evaluation
Results presented as means ± standard errors of the means were analyzed using one-way ANOVA and the Fisher protected least significant difference or by chi-square, when appropriate, with Statview Software (SAS Institute, Inc., Cary, NC) with statistical significance set at 0.05. Sample numbers in the databases for fertilization, development, pregnancy, and growth rates were influenced by experimental protocols and therefore vary.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Oocyte recovery from rhesus macaques has been described previously [4]. Recent experience involving 187 stimulations resulted in 41 (22%) cancellations because of failure to reach threshold expectations during stimulation (
3 follicles 4-mm diameter on each ovary). From 146 aspirations, the total number of oocytes recovered per animal was 38 ± 2, of which 22 ± 2 were MII at collection or within 4 h of collection and 8 ± 1 were GV intact. The balance was represented by MI oocytes that did not mature in vitro or atretic oocytes.
Fertilization and Embryo Culture
The IVF rate with fresh sperm in 2002–2003 was 61% ± 6% for 23 replicates involving 242 ova; fresh-sperm ICSI during the same time frame was 81% ± 3% for 61 replicates and 714 ova.
In general, conventional IVF is not feasible with frozen-thawed sperm because of the rapid motility loss after thawing. With the cryopreservation method described herein, motility declined from 30% to 5% within 4 h of thaw. Theoretically, this motility loss is not a problem when fertilization is by ICSI and motility is used only as a measure of sperm viability. In initial efforts to employ cryopreserved sperm for fertilization by ICSI, injections were conducted soon after sperm thaw and processing in an effort to select progressively motile sperm. However, the resultant fertilization rate after ICSI with frozen-thawed compared with fresh sperm was significantly reduced [22]. In a retrospective analysis, a relationship between time postthaw and fertilization by ICSI has emerged with low initial fertilization levels increasing to maximum levels after 3 h of sperm preincubation (Fig. 1). Thus, control fertilization rates equivalent to those achieved with ICSI using fresh sperm resulted when ICSI with cryopreserved sperm was performed at least 3 h after thawing. For the 2002–2003 season, an 80 ± 2% fertilization rate was achieved following ICSI with frozen-thawed sperm in 115 replicates involving 1473 ova.
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A complex medium, based on CMRL 1066 supplemented with serum, has historically been used to culture NHP embryos with or without coculture on BRL cell monolayers [CMRL/BRL; 5, 23, 24]. In 2002, we began the use of HECM-9 in the absence of coculture and have now evaluated 1715 oocytes. A 76% ± 2% ICSI fertilization rate was achieved with a 47% ± 4% blastocyst formation rate. Perhaps more significant than the number of embryos that reached the blastocyst stage in culture was the rate of progression. Culture in HECM-9 produced faster development than that seen in CMRL/BRL or KSOM-AA [25]. The average time to reach the morula stage (N = 222) was 4.7 ± 0.2 days, for blastocyst (N = 83) 6.4 ± 0.2 days, and for expanded, or hatched blastocysts (N = 78) 7.2 ± 0.3 days. While we have not conducted a controlled prospective trial measuring pregnancy outcome for embryos produced in these two culture systems, a retrospective analysis indicated that pregnancy and implantation rates were identical when ET was conducted with early stage (Days 2–4), cleaving embryos. For CMRL/BRL-produced embryos, 24 pregnancies were obtained in 96 transfers (25%), and for HECM-9-produced embryos, 20 pregnancies resulted from 77 transfers (26%).
Embryo Transfer
A number of parameters are known to contribute to pregnancy and implantation rates, including the number of embryos transferred, embryo quality, and the method of transfer. The impact of each of these was evaluated retrospectively.
High-order multiple pregnancy has been a problem in human clinical IVF secondary to the transfer of relatively large numbers of embryos. In monkeys, twinning is rare in nature and outcomes of twin pregnancies are problematic [21], such that we have sought to maximize singleton pregnancy rates without producing twins except for efforts to generate genetically identical animals. Pregnancy rates were equivalent or increased when the number of embryos transferred into each recipient was increased from 1 to 2 to 3 (Table 1). Interestingly, implantation rates were independent of the number of embryos transferred and the twinning rate did not increase when the number of embryos transferred went from 2 to 3.
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The selection of embryos for transfer is normally based on developmental progression, presence of the appropriate number of nucleated blastomeres, absence of fragmentation, and general appearance. Usually, only the highest quality embryos are transferred. The ability to freeze embryos and conduct transfers when recipients are available, while highly convenient, as it also supports the shipment of embryos to other facilities, carries the possibility of adversely affecting embryo quality. In our experience, pregnancy and implantation rates for fresh and frozen-thawed embryos were similar (Table 2). However, while the average number of embryos transferred was similar for fresh (2.3) and frozen (2.1) embryos, substantial embryo loss occurred with cryopreservation. Based on an evaluation of 304 frozen-thawed embryos, a cryosurvival rate of 65% was realized.
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Three different approaches to embryo transfer have been employed, mini-laparotomy, laparoscopy, and cervical cannulation. The first two methods result in oviductal deposition of embryos, while the latter involves uterine placement that is often precluded by the long, tortuous nature of the cervix in this species. Similar pregnancy and implantation results were obtained with both oviductal techniques that were significantly superior to the disappointing results obtained with the transcervical approach (Table 3). The proportions of intact and demiembryos were similar among treatments such that removal of demiembryos from the comparison did not affect the conclusion.
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Pregnancy success depends on the synchrony between the age of the transferred embryos, as measured by culture time in vitro, and the host endometrium relative to the predicted day of ovulation. Synchronous transfers appear to result in higher pregnancy rates in women. However, some evidence supports an asynchronous transfer in the rhesus monkey [19, 26], with optimal timing for blastocyst (Day 7 or 8) transfer into a Day 4 uterine environment. In contrast, younger cleavage-stage embryos might be transferred more appropriately into synchronized recipients. The low success rate following transcervical embryo transfer (7%; Table 3) led to a focus on results generated from oviductal transfers with intact or demiembryos only (Table 4). One-day-old embryos were transferred into a total of 27 recipients at four times postovulation, yielding an overall pregnancy rate of 15%. Transfers with Day 2 and Day 3 embryos led to pregnancy rates of 22% and 37%, respectively. Overall pregnancy rates did not vary significantly as a function of recipient menstrual age. Thus, the highest success rates were associated with the transfer of Day 3 embryos into either Day 2, 3, or 4 recipients. For embryos older than Day 5, while our experience is limited, the best results have been obtained following transfer into a younger recipient (Day 4; results not shown).
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Pregnancy and Pregnancy Outcome
With ICSI-produced embryos, 47 pregnancies resulted from 206 embryo transfers (23%) including successes with cryopreserved embryos shipped to other facilities. For twinned demiembryos, an overall 19% pregnancy rate was realized (14/72), including all methods of embryo transfer and involving both blastomere separation and blastocyst splitting [18]. The outcome of ART pregnancies as a function of the methods employed in embryo production was determined for both singleton and twin pregnancies (Table 5). A cohort of pregnancies from the TMB colony was included for comparative purposes in an effort to assess possible ART-related effects. In total, 234 of 257 (91%) clinical pregnancies resulted in live birth. In the TMB population, 14 cases of fetal demise were recorded, for a 92.5% live-birth rate. For ART-produced embryos (NT, demiembryos, ICSI, and IVF combined) 63 of 72 singleton pregnancies resulted in a live-birth rate of 87.5%, which was not significantly different from the TMB colony. When the ARTs were confined to ICSI and IVF, a combined live-birth rate of 92.8% (52/56) was observed. In contrast, singleton pregnancy outcomes resulting from demiembryo transfers were significantly reduced to a live-birth rate of only 69% (9/13).
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The twinning rate varied from zero in the timed mated breeding colony to a high, in the 17–21% range, for IVF or ICSI fertilized cases, when multiple embryos were transferred (Table 5). In total, there were 14 fraternal sets and 1 presumed identical set of twins in 87 pregnancies following application of the ARTs (17% of pregnancies). Eleven of these 15 twin pregnancies resulted in the birth of at least one viable fetus: 7 with 2 and 4 with 1. Thus, of the 30 fetuses, 18 were live term births, while 12 were lost, 5 as SB and 7 as SABs.
Birth weights of singleton, live term infants have been summarized in Table 6. The average singleton birth-weight for 84 TMB animals was 0.48 kg and for 61 ART-produced animals, 0.49 kg. The overall sex ratio was 73 female and 72 male, with no significant group differences between ARTs (32 female, 29 male) and TMB (41 female, 43 male; P = 0.7). Overall, male singleton infants were slightly heavier at birth with slightly longer gestational ages. A shorter gestational age was associated with ART infants (163.5 days for ICSI, IVF, and TW combined in Table 6 versus 166.6 for TMB; P = 0.013). A plausible explanation for a decreased gestational age could be an increased cesarean-section rate. This rate in the TMB colony was just under 3%, while for ART-related pregnancies, it was 27%. However, when comparisons were made for only those infants delivered vaginally, there was still a significantly shorter gestational age associated with ART pregnancies.
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In contrast, the average twin birth weight at 0.35 kg was significantly reduced in comparison with singleton infants. An average gestational age of only 150.7 days was also significantly shorter than that seen with singleton pregnancies. Four twin sets were delivered by cesarean-section and three vaginally without assistance.
Growth Rates
Animal growth rates based on weight for ART-produced singleton and twin pregnancies as well as singletons resulting from the TMB colony are summarized in Table 7. No differences were noted when singleton ART-produced infants were compared with TMB colony infants. However, low birth weight twins did not fall within the singleton weight range until 1 yr of age.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Handling and capacitation of ejaculated rhesus monkey sperm are also different from that of most species. On the positive side, samples collected by penile electroejaculation are of high quality, with uniform sperm morphology and few contaminating cells [16]. While simple removal of seminal plasma seems to be all that is required in preparation for human IVF, chemical capacitation has been described in the monkey, involving dibutyrylcyclic adenosine monophosphate and caffeine exposure [6]. A problem exacerbated by this treatment is a tendency for washed, capacitated sperm to agglutinate head-to-head to such an extent that successful IVF is jeopardized. The use of exceptionally high concentrations of motile sperm (0.5–1 x 106/ ml) has evolved as a means of overcoming this limitation. Alternatively, ICSI can be employed without chemical capacitation, an essential approach in the case of frozen-thawed sperm, where rapid motility decline occurs postthaw. In the latter case, our results showed optimized fertilization rates, i.e., comparable with IVF or fresh sperm ICSI, only after sperm incubation for 3 h postthaw. Because there was no indication that ICSI failure at the earlier time points was egg related, a plausible explanation for the required time delay involves sperm recovery from sublethal damage or restoration of the sperm's oocyte-activating properties [27].
Another unique problem with the ARTs in monkeys is embryo transfer. The rhesus macaque has a long, tortuous cervix that is difficult to cannulate [28, 29]. Even with careful animal preselection, the transcervical approach occasionally fails and, as reported here, pregnancy rates are unacceptably low. Endometrial trauma from cannula placement using only tactile palpation may contribute to the low pregnancy rates. However, implementation of progressively more elaborate methods, including ultrasound-guided catheter placement and cannula placement only to the level of the internal cervical os, did not result in improvement. Also, posttransplantation examination of the catheter for evidence of blood, as an indicator of possible endometrial trauma, could not be correlated with success rate. The development of an alternative, minimally invasive approach based on laparoscopy, resulted in pregnancy rates comparable with surgical introduction of embryos into the oviduct. This achieves the objective of minimizing animal exposure to major surgery. Other factors that must be considered in the success rate for both the transcervical and laparoscopic methods include catheter and cannula diameters. In both methods, embryo transfer catheters were designed for human applications. Thus, the larger size relative to the oviducts, despite the ease with which they were placed, may have had a negative effect on success rates. The use of smaller embryo-transfer catheters and further reducing possible oviductal trauma are future goals.
The pregnancy rate of 36% achieved in this study when three intact embryos were transferred and a live-birth rate of 33% compares favorably with a reported live birth/delivery rate in young women (
35 yr) of 32% when 3.5 embryos were transferred [30]. Because these monkeys are not experiencing recognized fertility problems, further improvements in pregnancy rates are realistic. Oocyte quality may be suboptimal [19], as we force follicular stimulations into a preset temporal schedule rather than individualizing this process, so refinement in this approach may be worthwhile. Improvements in culture conditions are, of course, possible, predicated on the extensive refinements that have occurred in the human ARTs. With regard to the timing of embryo transfer, synchrony issues now seem resolved, with close synchrony (Day 3 embryos into Day 2, 3, or 4 recipients) providing optimal results. Based on the finding that the twinning rate did not increase when the number of embryos transferred was increased from two to three, we are currently conducting a trial with four embryos/transfer. The plateau in the twinning rate seen here and in nature may reflect factors other than embryo number in determining multiple pregnancies. Thus, the rhesus macaque does not seem to be subject to high-order multiple pregnancies, as are women.
Our retrospective review of pregnancy outcomes showed that the fetal loss rate in IVF- or ICSI-related pregnancies was comparable with that seen in an indoor-housed TMB colony at under 10%. In caged and uncaged rhesus monkeys maintained at the California National Primate Research Center [21], total prenatal mortality was 17% (226/ 1332) and 16.4% (614/3736), respectively. The twinning rate was 0.1%, with poor pregnancy outcomes. With the ARTs, we report a twinning rate following transfer of >1 embryo of 17% with a net increase in the number of live infants produced; 15 pregnancies produced 18 infants. However, twinning is accompanied by undesirable sequalae from a maternal health perspective. First, there is a tendency to deliver twins by cesarean-section, meaning that a surgical procedure is involved and, second, the dam does not always care for the infants or cannot handle both of them postrecovery.
Based on observed birth weights for ART-produced pregnancies, there is no suggestion of a large-offspring syndrome in the rhesus monkey [31]. In fact, the slightly decreased birth weights compared with TMB infants is similar to the finding of lower birth weights in ART children [10]. Singleton infants originating from demiembryos showed normal birth weights, reflecting an inherent ability of the fetus to recover from the reduced number of cells at the time of embryo transfer. In contrast, twin pregnancies resulted in smaller infants delivered early. Our limited results suggest, however, that twins fall within the growth statistics for ART-produced infants by 12 mo of age.
As summarized in this report, the ARTs in rhesus monkeys now represent a feasible approach to producing animals. Cost-effectiveness issues should also be considered. Clearly, if there is no other way to acquire specific animals, i.e., they are priceless, then a discussion of this subject is irrelevant; Indian-origin, A*01 rhesus monkeys are perhaps an appropriate example [1]. Additionally, it is clear that the use of the ARTs will incur added charges over sheltered group housing where the cost of producing a 1-yr-old animal, based solely on 2.5 yr of per-diem maintenance charges, is approximately $2000 at this institution (0.5 yr for the father, 1.5 yr for the mother, and 0.5 yr for the infant). The comparable cost in a TMB colony jumps to $5000, reflecting the increased per-diem rates associated with individualized housing. In one of the least complex uses of the ARTs, artificial insemination, cycle determination by endocrine monitoring is required along with semen acquisition from an appropriately trained male; however, the cost per infant may only be modestly increased to approximately $6000. For IVF or ICSI, the animal needs expand further to include an oocyte donor and an embryo transfer recipient (three transfers required per live birth). Cycle monitoring must be conducted in several animals and a source of hormones for follicular stimulation must be identified, raising the overall cost per infant into the $7500–9000 range. These estimates assume that the technical support staff and the resources are in place to support the ARTs. The logistics of providing such services to the wider NHP research community could involve established centers that would use frozen sperm, conduct ICSI, and send out frozen embryos. Alternatively, the technology is readily transferable, requiring only access to an appropriately trained and staffed embryology unit possessing the relevant equipment.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Don P. Wolf, Oregon National Primate Research Center, 505 N.W. 185th Ave., Beaverton, OR 97006. FAX: 503 533 2494; wolfd{at}ohsu.edu
Received: 26 November 2003.
First decision: 22 December 2003.
Accepted: 16 March 2004.
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