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Biology of Reproduction 66, 1449-1455 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Regular Article

Monozygotic Twinning in Rhesus Monkeys by Manipulation of In Vitro-Derived Embryos1

Shoukhrat M. Mitalipova, Richard R. Yeomana, Hung-Chih Kuoa, and Don P. Wolf2,a

a Oregon Regional Primate Research Center, Oregon Health & Science University, Beaverton, Oregon 97006


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The nonhuman primate is a relevant model for human disease that can be used for diverse biomedical investigations. The ability to propagate a founder animal by application of assisted reproductive technologies is pressing, but an even greater need in many studies is access to genetically identical animals. In an effort to create genetically identical monkeys, we evaluated two approaches to monozygotic twinning; blastomere separation, and blastocyst bisection. Embryos were produced by intracytoplasmic sperm injection of oocytes recovered following controlled ovarian stimulation. The quality of demiembryos produced in these efforts was evaluated by quantitating the efficiency of creating identical pairs for embryo transfer, by morphological assessment, by the allocation of cells to the inner cell mass (ICM) and trophectoderm (TE) in the blastocyst, and by the outcome of embryo transfer to synchronized host animals. Pairs were produced in high yield (85%–95%) by both twinning methods. Demiembryos resulting from blastomere separations at the 2- or 4-cell stage grew to blastocysts at the control frequency. Demiblastocysts contained, on average, half the number of cells of the intact controls while maintaining the same ICM:TE or ICM:total cell ratio. The equivalency of demiblastocysts within a set was also evaluated by differential cell counting. Embryo transfers of identical sets led to a 33% clinical pregnancy rate, with two twin pregnancies initiated. Neither pregnancy resulted in term birth of monozygotic twins, but our results are sufficiently encouraging to justify a large-scale twinning trial in the rhesus macaque.

assisted reproductive technology, embryo, fertilization, in vitro fertilization, pregnancy


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The nonhuman primate is an important model for human disease that can be used for biomedical investigations of infectious diseases, including vaccine development, drug and alcohol addiction, transplant biology, brain function, and gene therapy. Currently, the significant need for populations of animals with specified genotypes cannot be satisfied by importation of animals from the wild or by identification and propagation of founder animals by selective breeding. Because they provide the opportunity to understand the mechanisms of protective immunity, Indian-origin rhesus macaques carrying the major histocompatibility complex (MHC) class 1 allele, A*01, are particularly needed for vaccine development, yet the wait for such animals is measured in years [1]. The ability to propagate a founder animal of defined MHC genotype is important, but an even greater need in the field of vaccine development or tissue transplantation, where immune system function is under study, is access to genetically identical animals. Somatic cell cloning may eventually provide a solution given the marked improvements being made in fetal and neonatal outcomes, although true clones (i.e., animals that are 100% genetically identical) will not result because of differences in the source of mitochondrial DNA [2]. The production of monozygotic offspring by manipulation of the preimplantation embryo remains an important alternative [3]. Two approaches have been established in several mammalian species: blastomere separation at very early developmental stages [3, 4], and blastocyst bisection [5]. Commercial interests in these technologies are based on the assumption that pregnancy and offspring efficiency can be enhanced by increasing the number of viable embryos transferred.

The ability of isolated blastomeres, either singly or in pairs, from 2-, 4-, and 8-cell stage embryos to support term pregnancies and to produce genetically identical animals has been described in the mouse [6], rat [7], goat [8], pig [9], horse [10], and, on repeated occasions, in cattle [5, 11, 12]. Although the usual outcome in the production of monozygotic offspring is singletons or twins, triplets and even quadruplets have been reported from transfer of one-quarter embryos in cattle [12, 13].

The splitting of uterine-stage embryos (i.e., morulae/blastocysts) has also led to the production of monozygotic offspring with commercial application. Embryos can be recovered nonsurgically by flushing the uterus of mated animals or by applying assisted reproductive technologies [5, 14, 15]. Using these approaches increased overall production of cattle following transfer of bisected embryos has been reported [16] based on a large number of transfers; moreover, even frozen-thawed demiembryos have been employed [17]. Morulae/blastocyst bisection has also been reported in the mouse [18, 19], rabbit [20], and pig [21, 22].

The production of identical rhesus monkeys by embryo manipulation has not been successful despite preliminary efforts [23–25]. Recovery of in vitro-derived embryos by uterine lavage is possible in this species [26, 27]; however, routine application of this approach is negatively impacted by high costs and low efficiencies. Fortunately, the infrastructure and experience in this area are now sufficient to allow routine production of embryos in vitro with culture to the blastocyst stage, low-temperature storage, and nonsurgical transfer [28], thus making it possible to contrast different approaches to the production of genetically identical monkeys. In this report, we describe the production of viable demiembryos from both blastomere separation and blastocyst bisection along with the limitations experienced in efforts to produce monozygotic twins.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

Mature rhesus macaque males and females housed in individual cages were used in this study. All animal procedures were approved by the Institutional Animal Care and Use Committee at the Oregon Regional Primate Research Center/Oregon Health & Science University.

Ovarian Stimulation and Recovery of Macaque Oocytes

The methods for rhesus monkey ovarian stimulation and oocyte recovery have been described previously [29]. Briefly, female rhesus macaques were subjected to follicular stimulation using twice-daily i.m. injections of recombinant human FSH as well as concurrent treatment with Antide, a GnRH antagonist, for 9 days [29, 30]. Females received recombinant human LH through Days 7–9 and recombinant hCG (rhCG) on Day 10. Unless indicated otherwise, all hormones were from Ares Advanced Technologies, Inc. (Norwell, MA), and other reagents were from Sigma-Aldrich Co. (St. Louis, MO).

Cumulus-oocyte complexes were collected from anesthetized animals by laparoscopic follicular aspiration (32–33 h post-rhCG) and placed in Hepes-buffered TALP (modified Tyrode solution with albumin, lactate, and pyruvate) medium [31] containing 0.3% (w/v) BSA (TH3) at 37°C. Oocytes, stripped of cumulus cells by mechanical pipetting after brief exposure (<1 min) to hyaluronidase (0.5 mg/ml), were placed in CMRL medium (Connaught Medical Research Laboratories; Invitrogen, Carlsbad, CA) at 37°C in 5% (w/v) CO2 containing 10% (v/v) fetal bovine serum (FBS; HyClone, Logan, UT), 10 mM L-glutamine (Invitrogen), 5 mM sodium pyruvate, 1 mM sodium lactate, 100 IU/ml of penicillin (Invitrogen), and 100 µg/ml of streptomycin (Invitrogen) until further use.

Collection and Processing of Sperm, Intracytoplasmic Sperm Injection, and Embryo Culture

Male rhesus macaques were trained to chair restraint and electro-ejaculated with a current isolation stimulator (P-T Electronics, Boring, OR) equipped with electrocardiographic pad electrodes for direct penile stimulation (30–50 V, 20-msec duration, 18 pulses/sec) as previously described [32, 33]. Semen samples were collected into 20-ml beakers, and after 15–30 min, an aliquot was taken and the sperm motility and concentration determined. The liquid portion of the ejaculate was diluted with 10 ml of TH3 and centrifuged for 5 min at 200 x g, after which the supernatant was removed. The pellet was resuspended in TH3, and the concentration was adjusted to 5 000 000 motile sperm/ml in TH3 and stored, on average, 3 h at room temperature before use.

Fertilization by intracytoplasmic sperm injection (ICSI) and embryo culture was performed as described previously [28, 34]. Briefly, sperm were diluted with 10% (w/v) polyvinylpyrrolidone (PVP; 1:4; Irvine Scientific, Santa Ana, CA), and a 5-µl drop was placed in a micromanipulation chamber. A drop of 30 µl of TH3 was placed in the same micromanipulation chamber next to the sperm droplet and covered with mineral oil. The micromanipulation chamber was mounted on an inverted microscope equipped with DIC or Hoffman optics and micromanipulators (Olympus America Inc., Melville, NY). An individual sperm was immobilized by striking the tail, aspirated into an ICSI pipette (Humagen, Charlottesville, VA), and injected into the cytoplasm of a mature metaphase II (MII) oocyte away from the polar body. After ICSI, embryos were placed in four-well dishes (Nalge Nunc International Co., Naperville, IL) and cocultured at 37°C in 5% CO2 on buffalo rat liver (BRL) cell monolayers in CMRL medium. Cultures were maintained under paraffin oil (Zander IVF, Vero Beach, FL). Pronuclear formation was recorded 16–20 h post-ICSI, and the progression of embryo growth was recorded daily. Embryos were transferred to fresh plates of BRL cells every other day for a maximum of 8 days.

Embryo Twinning by Blastomere Separation or Blastocyst Bisection

Surrogate zonae pellucidae were prepared from discarded monkey oocytes (nonmatured) by first making a slit in the zona with a glass microneedle and then removing the cytoplasm by aspiration with a micropipette. For blastomere separation, on Day 1–2 of culture, zonae pellucidae of selected 2- to 4- cell stage embryos were removed by brief exposure (45–60 sec) to 0.5% (w/v) pronase in Hepes-buffered TALP medium. Zona-free embryos were exposed to 0.5 mM EDTA in Ca2+- and Mg2+-free medium for 5 min to facilitate the separation of blastomeres and then transferred to TH3 containing 5 µg/ml of cytochalasin B. Individual (from 2-cell embryos) and paired (from 4-cell embryos) blastomeres were separated mechanically, aspirated into a micropipette, and transferred into empty zonae pellucidae immobilized on a holding pipette (Fig. 1). The resulting demiembryos were rinsed three times in TH3 and placed in coculture.



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  		FIG. 1. Embryo twinning by blastomere separation. A) 2-Cell stage rhesus monkey embryos after removal of zonae pellucidae. B) A single blastomere in the transfer pipette, positioned for placement inside a surrogate zona pellucida immobilized on a holding pipette. C) Monozygotic 1-cell demiembryos produced by blastomere separation. D) Hatching monozygotic demiblastocysts after in vitro culture for 8 days

For blastocyst bisection, mature oocytes were fertilized by ICSI and cultured to the blastocyst stage for 6–7 days as described above. The blastocyst was placed in TH3 medium and immobilized with a holding pipette positioned across from the inner cell mass (ICM) in a micromanipulation chamber on the stage of an inverted microscope. A surgical microblade (Accurate Surgical & Scientific Instruments, San Diego, CA) attached to a micromanipulator was used to split the embryo, with even distribution of ICM and trophectoderm (TE) into each demiembryo (Fig. 2). The zone-free demiembryos produced by bisection were placed in coculture until Day 8 and monitored for re-expansion.



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  		FIG. 2. Embryo twinning by blastocyst bisection. A) Expanded rhesus monkey blastocyst immobilized with a holding pipette positioned opposite the ICM. B) Initial bisection of the ICM with a surgical microblade while holding the blastocyst. C) Completion of the bisection process after release from the holding pipette. D) Monozygotic demiblastocyst immediately on completion of the bisection step

The development of demiembryos produced by blastomere separation or blastocyst bisection as well as the development of control, intact embryos was assessed daily by direct microscopic observation.

Differential Staining and Cell Count of Intact Blastocysts and Demiblastocysts

The nuclei of Day 8 demiblastocysts and intact blastocysts were differentially stained to determine the number of cells in the ICM and TE according to the procedure previously reported for human embryos [35] with slight modifications. Briefly, zona-free blastocysts were incubated in 10 mM trinitrobenzenesulphonic acid in CMRL medium containing 4 mg/ml of PVP on ice for 10 min, washed three times in TH3, and incubated a further 10 min in 0.1 mg/ml of rabbit antiserum against dinitrophenol, covalently linked to BSA (anti-DNP-BSA; ICN Biomedicals, Costa Mesa, CA), in Hepes-buffered TALP medium at 37°C. After three washes in TH3, blastocysts were incubated in a 1:10 dilution of guinea pig complement in Hepes-buffered TALP medium containing 2.5 µg/ml of propidium iodide (PI) at 37°C for 15–20 min. Then, embryos were washed in cold 85% (w/v) ethanol and fixed in absolute ethanol containing 50 µg/ml of bisbenzimide (Hoechst 33342) at 4°C for 1 h and washed again in 85% ethanol for 30 min. Differentially stained blastocysts were individually mounted on microscope slides in Vecta-shield (Vector Laboratories, Inc., Burlingame, CA) and covered with coverslips using spacers (thickness, 0.2 mm). Blastocysts were imaged with a Leica TCS SP (Leica Microsystems, Heidelberg, Germany) confocal microscope using 40x PlanApo NA 1.25 or 25x PlanFluotar NA 0.75 objectives. A 361-nm Ar laser and a 568-nm Kr laser were used to excite Hoechst 33342 and PI, respectively. Fluorescent light in the spectral range of 420–490 nm and 595–670 nm was detected simultaneously in separate channels for PI and Hoechst stain, respectively. Optical sections approximately 0.5 µm in thickness were sampled at 1- to 5-µm intervals throughout the thickness of the blastocyst. The PI and Hoechst 33342 optical sections were combined into three-dimensional (3D) images and analyzed using MetaMorph 4.5 (Universal Imaging Co., West Chester, PA). The average number of ICM and TE cells was calculated based on manual independent counts of the same specimen by two persons.

Embryo Transfer

Adult, multiparous females monitored for mense were used as recipients. Daily blood samples were collected beginning on Day 8 of the menstrual cycle, and serum levels of estradiol were quantitated by RIA. The day following the peak in serum estradiol was considered to be the day of ovulation (i.e., Day 0). Within 0–5 days of ovulation, recipient females were anesthetized with ketamine (10 mg/kg body weight i.m.; Fort Dodge Laboratories, Fort Dodge, IA) and prepared for embryo transfer. Typically, two embryos were transferred at minilaparotomy to the oviducts of recipients as described previously [32, 33]. Briefly, embryos were transferred to TH3 medium and loaded into a silicon tubing catheter controlled by a 1-cc syringe. The tip of the catheter was inserted (depth, 2–3 cm) into the oviduct, and the embryos were expelled. To detect pregnancy, serum levels of estrogen and progesterone were monitored every third day after embryo transfer. Pregnancy was confirmed by ultrasound approximately 25 days posttransfer and was monitored periodically throughout gestation.

Statistical Analysis

Results, expressed as the mean ± SEM, were analyzed using one-way ANOVA and the Fisher protected least significant difference test or the chi-square test when appropriate within Statview software (SAS Institute, Inc., Cary, NC).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Two methods for producing monozygotic demiembryos were evaluated by quantitating the efficiency of creating identical pairs for embryo transfer, by morphological assessment and differential cell counts of demiembryos, and by the outcome of embryo transfer. Blastomere separation was studied from 1999 to 2001 and blastocyst bisection from 2000 to 2001. The follicular stimulation protocol was initiated 42 times in a total of 31 animals. After follicular aspiration, an average of 39 ± 4 oocytes was recovered per stimulation, of which 20 ± 2 were classified as MII. Of the remaining oocytes, 10 ± 1 were metaphase I, of which 5 ± 1 matured to MII in culture within 3–4 h of collection. Oocytes collected from four stimulations (10%) were fertilized by ICSI but failed to develop to the blastocyst stage and, therefore, were removed from data analysis.

A critical measure of feasibility in monozygotic twinning is the efficiency of producing demiembryo pairs for embryo transfer. When blastomere separation was employed, the efficiency of producing demiembryos was greater (P < 0.01) at the 4- to 8-cell stage (38 embryos separated into 75 demiembryos, or 75 of a potential 76 [75/76]; 99%) compared to the 2-cell stage (62 embryos produced 110 demiembryos, or 110/124; 89%). Losses at the 2-cell stage occurred when aspirating these relatively large blastomeres into a micropipette in preparation for transfer into a surrogate zona. As many as 30% of cleavage-stage embryos were deemed to be unsuitable for blastomere separation due to uneven cleavage (one large and one small blastomere at the 2-cell stage) or the presence of an odd number of blastomeres (3- or 5-cells). The recovery of pairs of genetically identical demiembryos from those embryos that were manipulated was 85% (85/100).

Blastocyst bisection was slightly more efficient in the creation of demiembryos (76/80; 95%) and in the recovery of pairs (36/40; 90%). It is interesting that the much more invasive surgical bisection was less prone to embryo demise than was teasing blastomeres apart at early cleavage stages.

In the case of blastomere separation, a convenient measure of demiembryo viability is growth to the blastocyst stage in vitro (6–8 days of culture) compared with intact, ICSI-produced controls. Of 72 intact embryos, 50 (69%), 39 (54%), and 25 (35%) developed to the morula, compact morula, and blastocyst stage, respectively. When an identical number of demiembryos was cultured, 55 (76%), 54 (75%), and 34 (47%) reached the morula, compact morula, and blastocyst stage, respectively. The control blastocyst development rate (36%) may be artificially low, because the highest-quality embryos were set aside for twinning [28]. Nevertheless, no significant difference was observed in the developmental competence of demiembryos produced by blastomere separation when compared to intact controls.

In the case of embryo bisection, in vitro culture to the stage for manipulation is, of course, 100%. In preliminary experimentation, bisection at the compact morula stage was eliminated from consideration, because only 1 of 12 demiembryos survived and continued development to the blastocyst stage (results not shown). In contrast, when bisecting blastocysts, 76 of 80 demiembryos survived; four demiembryos stuck to the knife and were lost during the procedure.

Differential Staining of Demiembryos

The number of TE and ICM cells was quantitated in control embryos and in demiembryos produced by blastocyst bisection or blastomere separation (Table 1). Demiembryos produced by the two approaches were statistically similar by all measured parameters. The averaged total cell number in demiembryos (n = 132) was 51% of the 258 cells counted in the intact control. Demiembryos were comprised of an average of 39 ICM cells (42% of control) and 93 TE cells (56% of control). The ICM:TE and ICM:total cell ratios were also statistically the same for all embryos.


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  		TABLE 1. Cell count of Day 8 ICSI-produced demiblastocysts and control, intact blastocysts of the thesus monkey

In evaluating the equivalency of demiembryos within a set of twins, cell counts for matched pairs have been presented separately (Table 2). The TE and ICM counts on member a versus member b for demiembryo pairs produced by blastomere separation appear in the top section of Table 2. In these experiments, blastomere separation at the 2-cell stage was followed by 5 days of culture and differential staining at the blastocyst stage. As can be seen, one member of each demiembryo pair was quite different from the other; for instance, total cell numbers of 53 and 217 for one set and of 105 and 162 for the other. The ICM:total cell ratio varied from a low of 0.12 to a high of 0.38.


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  		TABLE 2. Cell count of Day 8 demiblastocyst pairs produced by embryo manipulation in the rhesus monkey

For demiembryo pairs created by blastocyst bisection, five pairs were analyzed (Table 2, bottom section). A relatively large variation was found in total cell counts (e.g., 43 and 57 for pair 5 versus 220 and 171 for pair 2), but the counts within a set of twins were similar. The ICM:total cell ratios varied from a low of 0.21 and 0.26 for pair 2 to a high of 0.51 and 0.53 for pair 5. These results support the conclusion that the bisection process creates demiembryo pairs of similar cell number and distribution.

In addition to differential cell counts at the blastocyst stage, we compared embryo morphology at the stage of development when demiembryo sets were transferred to recipients or were stored frozen. In the case of demiembryos produced by separation and evaluated between the 2-cell and the morula stage (i.e., at the time of transfer), 5 of 21 pairs (24%) were at the same stage. In those twin sets that were retained in culture and reached the blastocyst stage, 10 of 16 pairs (63%) were at the same stage. In contrast, when blastocyst bisection was used to produce demiembryos, 8 of 10 pairs (80%) were at the same developmental stage after one additional day of culture.

In Vivo Developmental Potential of Demiembryos

Transfer of 44 embryos (22 sets of identical pairs) produced by blastomere separation into recipients led to a 33% pregnancy rate (7/22), with two twin pregnancies initiated. However, one twin pair was spontaneously aborted at 75 days of gestation, and in the second, one member of the pair was resorbed spontaneously, leaving a singleton pregnancy. With bisected blastocysts, a 33% pregnancy rate was also obtained (4/12), with all pregnancies being singletons. Of these, 16 bisected blastocysts were transferred as identical pairs into eight recipients (two pregnancies; 25% clinical pregnancy rate), and four bisected blastocysts (two pairs) were transferred singly into each of four recipients (two pregnancies, one from each pair; 50% clinical pregnancy rate).

The pregnancy success rate as a function of the timing between the demiembryo(s) transferred, as measured by culture time in vitro, and the host endometrium, relative to the predicted day of ovulation (1 day after the peak estrogen level), is itemized in Table 3. No pregnancies were obtained when transfers were conducted into Day 0 or Day 5 recipients. Optimal results (3/5; 60%) for blastocyst (Days 7 and 8) transfer were obtained into a Day 4 uterine environment (i.e., asynchronous transfer). In contrast, demiembryos produced by blastomere separation and transferred at a culture age of 2–6 days were optimally transferred into a Day 2 host, and again, the peak pregnancy rate was 60%.


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  		TABLE 3. Relationship in age between embryo (culture time in days) and recipient (days postovulatory) in surgical demiembryo transfers conducted in the rhesus monkey

The outcomes of these pregnancies are summarized in Table 4. Also included in Table 4, for comparative purposes, are the historical results with pregnancies obtained by this laboratory from intact embryos, either derived by ICSI or after conventional insemination and embryo cryopreservation. No differences were apparent in birth weights following singleton pregnancies derived from demiembryos versus those of the ICSI-produced, intact controls. Eight of the nine pregnancies in the in vitro fertilization (IVF) group were twin gestations, and the embryos had been cryopreserved before transfer [33].


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  		TABLE 4. Gestation length, gender, and birth weights of rhesus monkey infants produced by embryo twinning, ICSI, and IVE


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this report, we describe the application of simple embryo separation techniques used on rhesus monkey embryos with the long-term objective of producing monozygotic offspring. The advantage to producing identical animals for biomedical research is that, in general, a reduction in animal number requirements can be realized [36]. Additionally, genetically identical animals are absolute requirements for some experimentation, such as that involving immune system function.

The number of identical animals that can be produced by blastomere separation is realistically limited to twins or triplets, because the developmental potential of one-quarter embryos (and less) is poor secondary to inadequate cell numbers to support the allocation of cells required during blastulation, as evidenced by extensive experience in agricultural species [37]. Thus, embryonic mass may be insufficient for normal development or for the appropriate signaling that must occur between the embryo and host. Similarly, blastocyst bisection is realistically limited to monozygotic twins, because efforts to repeat the process have not been associated with high efficiencies [38]. Our blastocyst bisection results indicate that the blastocyst, not the morula, is the appropriate stage to split in the monkey, which is similar to a conclusion drawn in the pig [39].

In this study, demiembryos separated at the 2- or 4-cell stage grew to the blastocyst stage at the same rate as intact controls, and demiblastocysts, independent of the method of production, contained, as expected, half the number of cells, with ICM:TE or ICM:total cell ratios being the same as those of the intact controls. The ICM and ICM:total cell estimates obtained here for controls were consistent with those reported by us previously for Day 8 blastocysts: 305 ± 69 total cells, with ICM:total cell ratios of 0.39 [40]. In the present report, Day 8 blastocysts produced by ICSI contained 258 ± 25 cells, with ICM:total cell ratios of 0.37. Substantially lower numbers have been reported previously as determined by confocal imaging and 3D reconstruction: 136 cells, and ICM:total cell ratios of 0.10 for IVF-produced blastocysts [24]. Differences in the ICM:total cell ratios may reflect differences in methodologies, but why the total cell numbers should differ between studies, given similar embryo ages, is not clear. Nevertheless, cell count differences are presumably of minor concern when within-experiment comparisons are made between demiembryos and intact embryos.

When demiembryos were produced by blastomere separation and then cultured to the blastocyst stage before analysis, cell number differences within the two pairs examined were remarkable, suggesting that the separation process was detrimental despite resulting in demiembryos that were capable of growth. Suboptimal in vitro culture conditions, uneven distribution of cytoplasm between the two blastomeres at separation, or differences in polarity [41] may have accounted for these discrepancies. In contrast, demiembryo sets produced by blastocyst bisection showed little interset variability, and whereas we can conclude that these identical sets have similar characteristics, it is not yet possible to relate cell number or ratio information to in vivo developmental potential. Another difference between the demiembryos produced by the two methods concerns the presence of a zona. In bisected blastocysts, the zona was removed, whereas separated blastomeres were cultured with zonae. Early zona removal may impact developmental rates [37, 39] and the maintenance of cellular arrangements [42]. Cell allocation has been studied extensively in bisected pig embryos produced in vivo [39]. A low percentage of demiembryos was observed that contained no detectable ICM. However, in the majority of demiembryos, the ICM:TE ratio was relatively constant despite embryo-quality scores and splitting stages [39].

Based on the results presented here, preliminary estimates can be made regarding the efficiency of producing pregnancies by blastomere separation or blastocyst bisection in the monkey. Starting with 10 MII oocytes and assuming a fertilization level of 85% by ICSI, a blastocyst development rate of 50% and a clinical pregnancy rate of 40% from a single embryo transfer yields 3.4 and 1.7 clinical pregnancies per 10 oocytes with a Day 2–3 (early cleavage stage) and a Day 6–8 (blastocyst) embryo transfer, respectively. In the case of bisection, the number of available embryos would be increased by a factor of 1.8 (2 x 0.9 recovery of pairs), so the corresponding efficiency for a Day 6–8 demiembryo transfer would be 3.1 (10 x 0.85 x 0.5 x 0.4 x 1.8). With blastomere separation, the embryo-doubling stage is essentially the stage for transfer (85% recovery of pairs), and an efficiency of 5.8 clinical pregnancies per 10 oocytes results (10 x 0.85 x 0.85 x 2 x 0.4). However, if only 70% of early cleaving embryos are amenable to separation, this value would be reduced to 4.1. From an animal husbandry perspective, the surgical route of embryo transfer used for early cleavage stage embryos to the oviduct is decidedly inferior, considering the intervention, to the nonsurgical, transcervical approach that has been used with blastocysts [28]. The possibility, of course, remains that early cleavage stages could also be transferred nonsurgically into the uterus, analogous to the approach taken in human IVF [43].

Whereas we have shown production of demiembryos that implant at or near intact control rates and that produce term pregnancies, we were not successful in creating term pregnancies of monozygotic twins despite initiating two twin gestations. Fetal demise in the case of twin pregnancies is not unexpected in the monkey despite our previous successful twin births following the transfer of in vitro-derived, frozen-thawed embryos [33, 44]. Perinatal mortality in nonhuman primates caged indoors is approximately 15%, and with twins, which occur at a 0.25% frequency (7/2763), the majority of offspring fail to survive because of undefined complications in the fetal or perinatal period [45]. One difference between the current low success rates with twin gestations compared to our previous outcomes is the present use of Chinese-origin rhesus monkeys. Chinese-origin animals are slightly smaller than their Indian-origin cousins and may, secondary to their relatively recent captivity, be more susceptible to stress.

To further increase pregnancy and delivery rates, substantial development of infrastructure in this area remains to be done, including controlled ovarian stimulation protocols that yield more consistent and improved oocyte quality, better culture conditions, and higher implantation efficiencies. The timing of embryo transfer is also obviously of importance, and we previously described optimal pregnancy rates for in vitro-grown blastocysts when Day 8 embryos were transferred nonsurgically into a Day 4 uterine environment [28]. Consistent with these results, bisected blastocysts were also best-transferred asynchronously into a Day 4 uterine environment, and similar findings with in vivo-flushed embryos have been reported recently [46].

In summary, twinning methods applied to ICSI-fertilized embryos produced viable demiembryos that resulted in term pregnancies following asynchronous transfer. Blastocyst bisection was associated with a somewhat higher yield of monozygotic pairs for embryo transfer; however the estimated yield of clinical pregnancies per oocyte was higher for the separation procedure, which was not limited by the need to culture in vitro to the blastocyst stage. Demiembryos produced by these simple techniques have, on average, half the number of cells of their intact counterparts, maintain a similar allocation of cells to the ICM and TE, and show comparable implantation efficiency. These results are sufficiently encouraging to justify a large-scale twinning experiment evaluating outcomes when a set of monozygotic embryos is transferred to a single recipient as opposed to a single embryo transferred into each of two hosts.


   ACKNOWLEDGMENTS
 
We are grateful to Andrea Widmann-Browning, Cathy Ramsey, and Behzad Gerami-Naini for their technical assistance; Dr. John Fanton for laparoscopic oocyte retrieval; Dr. Anda Cornea for confocal microscopy; Julianne White for secretarial support; and Joel Ito for assistance with illustrative material. The Assisted Reproductive Technologies core facility of the Oregon Regional Primate Research Center assisted by providing semen samples. We would also like to acknowledge Ares Advanced Technology, Inc., a member of the Ares-Serono group of companies, for their generous donation of hormones used in this study.


   FOOTNOTES
 
First decision: 25 September 2001.

1 Supported by National Institutes of Health grant RR 12804 to D.P.W. Back

2 Correspondence: Don Wolf, Oregon Regional Primate Research Center, 505 NW 185th Ave., Beaverton, OR 97006. FAX: 503 690 5384; wolfd{at}ohsu.edu Back

Accepted: December 10, 2001.

Received: August 20, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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