Developmental competence of oocytes after ICSI in the rhesus monkey

K.D. Nusser1, S. Mitalipov1, A. Widmann1, B. Gerami-Naini1, R.R. Yeoman2 and D.P. Wolf1,2,3,4

1 Division of Reproductive Sciences, Oregon Regional Primate Research Center, Beaverton, OR 97006-3348 and Departments of 2 Obstetrics and Gynecology and 3 Physiology and Pharmacology, Oregon Health Sciences University, Portland, OR 97201-3098, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Note added in proof
 References
 
Oocyte quantity and quality are critical to assisted reproductive technology (ART), yet few assessments beyond counting metaphase II (MII) oocytes exist. In this study, 30 ± 2 oocytes per cycle were recovered from rhesus monkeys subjected to follicular stimulation with human gonadotrophins, of which 15 ± 1 were MII. Oocyte quality was investigated by monitoring the developmental potential of oocytes subjected to intracytoplasmic sperm injection (ICSI). Despite uniform fertilization rates (71 ± 4%), progression of embryos to blastocysts varied when expressed as a monthly average, from 20 to 85%, with lows from February to April and again in October, which could be attributed to developmental failure of a significant number of oocyte cohorts (14 of 55). Blastocyst rates, after elimination of failed cohorts, were uniform over time (59 ± 4%). Neither culture conditions, the number of follicular stimulations, nor the individual sperm or oocyte donor were associated specifically with developmental failure, suggesting that intrinsic differences between stimulation cycles account for the observed variation in developmental potential. The in-vivo developmental competence of ICSI-produced embryos grown to blastocysts in vitro was also assessed. Two ongoing pregnancies and the birth of a normal female, `Blastulina', represent landmarks in efforts to expand the use of ART in the rhesus monkey.

Key words: blastocyst/embryo transfer/ICSI/monkey/oocyte quality


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Note added in proof
 References
 
Any activity that requires the creation and development of an embryo, whether it is in the context of infertility treatment or in the creation of a reconstructed embryo by nuclear transfer, is dependent not only on oocyte quantity but also on the intrinsic ability of that oocyte to support development. Although limitations in oocyte quantity in non-human primates have been largely alleviated by the development of follicular stimulation protocols that result in the recovery of multiple oocytes (Wolf et al., 1990Go), animal usage—and hence oocyte recovery—is ultimately restricted by an immune response that renders the ovary refractory to repeated stimulation (Stouffer et al., 1983Go; Zelinski-Wooten et al., 1996Go).

Oocyte quality considerations are of unique concern in any procedures where efficiencies are low, since the intrinsic differences between oocytes cannot be separated readily from extrinsic factors. An evaluation of oocyte competence might reasonably be based on an assessment of the developmental potential of the resultant embryo. Confounding issues associated with the role of the spermatozoa and downstream events following oocyte activation detract from using fertilization as the sole measure of developmental potential. Disadvantages can also be associated with the use of in-vitro development to the blastocyst stage, since all blastocysts are not the same. For instance, parthenotes develop to blastocysts but do not support term pregnancies (Tarkowski et al., 1970Go), and significant differences in blastocyst quality have been defined clinically (Gardner and Schoolcraft, 1998Go). On the other hand, attainment of the blastocyst stage is a significant accomplishment, as the embryo—if normal and diploid—has progressed beyond the maternal to embryonic transition in the genomic control of development and has reached a differentiated stage with over 100 cells, a trophectoderm and an inner cell mass. The ultimate measure of embryo quality is unquestionably the ability to support term pregnancy following transfer to a suitable recipient. Unfortunately, it is often impractical to use term birth as the outcome measure because of time and resource considerations. In our ongoing efforts to improve the assisted reproductive technologies (ART) in support of nuclear transfer attempts in the rhesus monkey, we sought a system that would enable us to monitor the developmental competence of oocyte cohorts used for experimental purposes.

In the rhesus monkey, in-vitro developmental efficiencies of IVF-produced blastocysts range from a low of 8–22% (Boatman, 1987Go; Zhang et al., 1994Go) to a high of 66% (Weston and Wolf, 1996Go). However, this prior experience has been so limited that attainment of the blastocyst stage as an oocyte/embryo quality assessment measure was never considered. Even in embryos that develop to expanded blastocysts, in the absence of embryo transfer and pregnancy it must be recognized that they may be abnormal, secondary to inadequate inner cell mass development (Enders et al., 1989Go), and that pregnancy establishment may be impacted by embryo–uterine asynchrony.

Another consideration in devising a screening protocol for oocyte competence involves in-vitro fertilization. In the rhesus monkey, sperm capacitation and activation is a unique challenge. To achieve consistent fertilization, washed spermatozoa must be preincubated in albumin-containing medium followed by exposure to the activating agents, caffeine and dibutyryl cyclic AMP (Boatman and Bavister, 1984Go), which may act via protein kinase (Osheroff et al., 1999Go). Unfortunately, sperm activation is associated with an increased propensity for spermatozoa to agglutinate, which may impact survival as well as fertility. In order to avoid this potential rate-limiting step and minimize variability due to sperm capacitation and penetration, intracytoplasmic sperm injection (ICSI) was selected as the best inseminating technique for quality evaluation studies. The use of ICSI in humans is extensive from its original description in 1992 (Palermo et al., 1992Go) to its current use in 30% of all human ART cases (Society for Assisted Reproductive Technology, 1999). Typically, very high levels of fertilization are obtained with this technique in the human (Hsu et al., 1999Go). In the rhesus monkey, the participation of the sperm head in ICSI-induced oocyte activation has been documented (Meng and Wolf, 1997Go) and, recently, the first ICSI monkey was born following transfer of an 8-cell stage embryo (Hewitson et al., 1999Go).

Here, we summarize inclusive results from one year of activity, over parts of two experimental seasons, with regards to oocyte quantity and quality. For the latter, developmental competence was assessed by in-vitro embryo performance following fertilization by ICSI. Approximately 25% of oocyte cohorts were found to be developmentally incompetent; however, no obvious explanation for the poor performance could be defined. Additionally, we initiated a quality evaluation of in-vitro-produced blastocysts by conducting embryo transfers and report the first birth of a normal infant, `Blastulina', and two ongoing pregnancies following transfer of in-vitro-produced ICSI blastocysts.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Note added in proof
 References
 
Follicular stimulation and oocyte collection
Adult, female rhesus monkeys (n = 99) ranging in age from 4 to 17 years were used in the present study. Prior parity was documented in 63 of these animals. Animals were housed individually, and menses records recorded daily. At 1–4 days after menses, cycling females were entered into a follicular stimulation protocol (Zelinski-Wooten et al., 1995Go; Meng and Wolf, 1997Go). Briefly, monkeys received twice-daily injections of recombinant human FSH (rhFSH; 30 IU i.m.) and once-daily injections of Antide (a GnRH antagonist; 0.5 mg/kg s.c.) for 8–9 consecutive days. On the last 2 days of rhFSH/Antide stimulation, animals also received twice-daily injections of recombinant human LH (rhLH; 30 IU i.m.). On the last day of hormonal stimulation, ovarian morphology was recorded by ultrasonography (Advanced Technology Laboratories, ATL; Bothell, WA, USA). The size and number of follicles were noted, and the stimulation cycles in animals with follicles <3 mm diameter were cancelled and defined as non-responders. Monkeys responding to follicular stimulation received an injection of recombinant human chorionic gonadotrophin (rHCG; 1000 IU i.m.) to induce oocyte maturation. Cumulus–oocyte complexes (COC) were collected from follicular aspirates and placed in HEPES-buffered Tyrode's albumin lactate pyruvate (TALP) medium (Bavister et al., 1984Go) containing 0.3% bovine serum albumin at 37°C (BSA) (TH3 medium). Unless indicated otherwise, reagents were obtained from Sigma-Aldrich Co. (St Louis, MO, USA).

Oocytes were freed from the COC by mechanical trituration after brief exposure (1 min at 37°C) to hyaluronidase (1 mg/ml). Oocytes were then graded as to their maturational status (GV, containing a germinal vesicle; MI, lacking both a GV and the first polar body; or MII, containing the first polar body) and placed in Connaught Medical Research Laboratory (CMRL 1066) media (Boatman, 1987; Life Technologies, Rockville, MD, USA) containing 10% fetal bovine serum (FBS; HyClone, Logan, UT, USA), 10mmol/l L-glutamine (Life Technologies), 5 mmol/l sodium pyruvate, 1 mmol/l sodium lactate, 100 units/ml of penicillin (Life Technologies) and 100 µg/ml streptomycin (Life Technologies) until further use.

Animals were subjected to follicular stimulation and follicle aspiration as many as four times, and re-entered into the stimulation protocol only after experiencing a normal menses subsequent to their last surgical procedure (either follicular aspiration or embryo transfer).

ICSI protocol
After grading, a cohort of MII oocytes was subjected to ICSI (Meng and Wolf, 1997Go) within 4–5 h after surgical retrieval employing spermatozoa collected by penile electrostimulation from pregnancy-proven males (n = 9) (Lanzendorf et al., 1990aGo). Spermatozoa were prepared by centrifugation (7 min, 200 g) of the liquid portion of the ejaculate and resuspending the sperm pellet in TH3 medium. Before final centrifugation and resuspension, an aliquot was taken to determine motility and concentration. Sperm concentrations were adjusted to 5x106 motile spermatozoa per ml in TH3 medium and stored (on average) for 3 h at room temperature prior to ICSI.

An oil-covered micromanipulation chamber having a 30 µl TH3 drop for oocytes and a 4 µl drop of 10% polyvinyl pyrrolidone (Irvine Scientific, Santa Ana, CA, USA) for spermatozoa was prepared. Using an inverted microscope (Olympus IX70, Melville, NY, USA) with micromanipulators (Narishige, Tokyo, Japan), individual spermatozoa were immobilized, aspirated into an ICSI pipette (Humagen, Charlottesville, VA, USA) and injected into the oocyte cytoplasm, away from the polar body. When possible, in order to control for variations in culture conditions, ICSI was performed on oocytes from two separate donors.

Embryo culture
After ICSI, embryos were placed in 4-well Petri dishes (Nalge Nunc, Naperville, IL, USA) and co-cultured at 37°C in a 5% CO2 incubator with buffalo rat liver (BRL) cells (25 000 cells/well) in CMRL media supplemented with 10% FBS, as described above. Embryos were transferred to fresh plates of BRL cells every other day. Pronuclear formation was recorded 16–20 h post-ICSI, and the progression of embryo growth was recorded daily. Pronucleus formation and/or the timely cleavage of nucleated embryos were used as fertilization measures for oocytes subjected to ICSI. Morula and blastocyst rates were used to assess embryo development for each replicate, and were determined relative to the number of fertilized oocytes. Blastocyst formation was defined as the expansion of the embryo after compaction and cavitation to include both trophectoderm and inner cell mass.

Embryo transfer
Embryo transfers were conducted non-surgically, employing a transcervical approach with one exception, which employed a surgical method (Wolf et al., 1990Go). Adult, multiparous females were used as recipients and monitored for menses. Daily blood samples were collected beginning on day 8 after menses, and serum was measured for oestradiol by radioimmunoassay (Hess et al., 1981Go). The menstrual cycle stage of the recipient was determined by detection of the oestradiol surge, with the following day considered the day of ovulation (day 0). Between days 3 and 6 after ovulation, recipient females were anaesthetized with ketamine (10 mg/kg body weight, i.m.; Fort Dodge Laboratories, Fort Dodge, IA, USA), positioned in sternal recumbence and prepared for embryo transfer. Intrauterine embryo transfer was based on a previously described technique (Goodeaux et al., 1990Go). Briefly, a stylet was inserted into the vagina, using rectal palpation to guide placement through the cervix and into the uterus. A cannula was then placed over the stylet and positioned in the uterus, whereupon the stylet was removed. ICSI-produced embryos were transferred into TH3 medium and aspirated into the tip of a polyvinyl catheter connected to a 20 gauge blunt-tipped needle and a 1 ml syringe. The catheter was then inserted through the cannula into the uterus. The blastocyst(s) was then slowly expelled into the uterus and the catheter gently withdrawn. To detect pregnancy, serum concentrations of oestrogen and progesterone were monitored in blood samples taken from embryo recipients every third day after embryo transfer. Progression of the pregnancy was monitored periodically by ultrasonography.

Statistical analysis
Results were expressed as means (± SEM), and analysed using one-way ANOVA statistics and Fisher's PLSD (Protected Least Significant Difference) test within Statview software (SAS Institute Inc., Cary, NC, USA). Percentages were transformed by arcsin prior to analysis.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Note added in proof
 References
 
Follicular stimulation and oocyte recovery
The follicular stimulation protocol was initiated 174 times in a total of 99 animals. Of these protocols, 29 (16%) were discontinued due to lack of adequate response as judged by ultrasonography (follicles <3 mm diameter). The percentage of `non-responders' varied by season when expressed as a monthly average, and showed an increase during the summer months (Figure 1Go), reaching over 35% in June and September. A confounding factor that may have contributed to this increase in the non-response rate was the number of cycles of follicular stimulation experienced by the oocyte donor (Figure 1Go). Animals were typically assigned to the programme in the autumn such that most first time stimulation cycles occurred between October and February. As can be seen in Figure 1Go, the percentage of non-responders was highly correlated (P < 0.0001) to the number of stimulation cycles, with the highest non-response rate in June and September corresponding to peaks in stimulation cycle numbers approaching three. In addition to the lack of response to ovarian stimulation, another variable that impacted oocyte collection efforts was the regularity of menstrual cycling. During the summer, decreasing animal availability due to fewer females exhibiting normal menstrual cycles (Figure 2Go), in combination with an increase in non-responders among cycling females, resulted in the abandonment of follicular stimulation efforts in August.



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Figure 1. Percentage of rhesus monkeys (n = 99) classified as non-responders (<3 mm diameter follicles following follicular stimulation) as determined by ultrasound and the average number of stimulation cycles per animal. There were no non-responders in December, and no stimulations in August.

 


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Figure 2. Percentage of rhesus monkeys (n = 99) undergoing normal menstrual cycles over a 1-year interval.

 
From the 145 follicular stimulation cycles that resulted in follicular aspiration, an average of 30 ± 2 oocytes was recovered per stimulation, of which 15 ± 1 were classified as MII. Of the remaining oocytes, 8 ± 1 were classified as MI, 55% of which matured to MII in culture within 4 h of collection. In contrast to the changes over time noted in the percentage of menstruating or non-responding animals, the monthly average number of oocytes (total or MII) recovered from monkeys that responded to follicular stimulation was constant (Figure 3Go). Moreover, despite the increased non-response rate in females undergoing repeated stimulation, oocyte numbers in responding animals were not impacted by repeated exposure to human gonadotrophins (Table IGo).



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Figure 3. Average number per animal of total and MII oocytes recovered during the experimental season from rhesus monkeys subjected to follicular stimulation with exogenous gonadotrophins.

 

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Table I. Oocyte parameters in follicular stimulation cycles in rhesus monkeys
 
Fertilization and embryonic development
While the present study was designed as a prospective evaluation of oocyte developmental potential, other experimental objectives were often paramount, namely use of embryos as a source of blastomeres for nuclear transfer, for embryo splitting, or for embryo transfer. Therefore, of the 145 follicular stimulation cycles that went to oocyte collection, 90 cycles were eliminated from developmental studies due to inadequate oocyte numbers to support all activities. Within the 55 cycles included, an average of 6.8 ± 0.7 MII oocytes per replicate were allocated for ICSI and this number, by design, remained constant over time.

The fertilization rate per cycle within the 55 cycles, i.e. the percentage of ICSI oocytes that formed two pronuclei and/or cleaved into nucleated blastomeres, was 71 ± 4%. This fertilization rate, when expressed as a monthly average, was constant over time (Figure 4Go) and varied from a low of 53% to a high of 85%. In contrast to stable fertilization rates, the developmental potential (blastocyst rate) of these ICSI embryos, expressed as the percentage of fertilized oocytes that progressed to blastocysts, varied dramatically between individual cycles from 0 to 100%. When expressed as a monthly average, the blastocyst rate varied between 20 and 85% in a biphasic fashion, with lows from February to April and again in October (Figure 4Go). When cycles in which none of the embryos developed to blastocysts (blastocyst-incompetent cohorts) was removed from the analysis (14 of the 55 oocyte cohorts), average monthly blastocyst rates as anticipated (similar to the fertilization rate) became constant. Fertilization rates and the percentage of embryos reaching the morula stage were also different between developmentally competent and incompetent cohorts (P < 0.01), suggesting that discriminating end points occur prior to blastocyst formation (Table IIGo). In an effort to evaluate possible influences on oocyte developmental competence, culture conditions, prior ovarian exposure to human gonadotrophins, and the individual sperm and oocyte donor were considered.



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Figure 4. Fertilization and embryonic development rates of rhesus monkey oocytes/embryos observed during a 1-year interval. Fertilization, by intracytoplasmic sperm injection, was defined as the presence of two pronuclei and/or the timely cleavage of embryos into two or more nucleated cells. The blastocyst rate was defined as the percentage of fertilized oocytes that developed to blastocysts. Values which differ (P < 0.05) are denoted with differing letters.

 

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Table II. Summary of fertilization and oocyte development rates for blastocyst competent and blastocyst incompetent cohorts in the rhesus monkey
 
The complex co-culture system employed here may have impacted embryonic development secondary to the use of different lots of serum or differences in BRL cell passage number, cell density or cell viability. Parenthetically, efforts to employ more defined, sequential culture media such as that used clinically in humans have not resulted in consistent, high levels of embryonic development in the monkey. In an effort to evaluate the impact of culture conditions on embryonic development, ICSI was conducted (when possible) on oocytes from two donors and the resultant embryos were cultured separately but concurrently. Fifteen oocyte cohort pairs were cultured simultaneously and, of these pairs, nine included only developmentally competent cohorts, four included both a competent and an incompetent cohort, and two contained only developmentally incompetent cohorts. This outcome (nine competent, four competent and incompetent, and two incompetent) resembles the predicted outcome of eight, six and one, assuming that the 75/25% competent/incompetent rate is random (calculated from all experimental data). The poor association between developmental outcome and culture conditions, combined with the correlation of results to a predictable random event, suggest that the culture conditions are not a main factor in oocyte competence.

Another parameter with potential impact on oocyte developmental potential was the number of cycles experienced by the oocyte donor. As noted above, prior ovarian exposure had no effect on oocyte collection numbers (Table IGo), although multiple stimulations were associated with an increase in the non-response rate (Figure 1Go). In Table IIIGo, fertilization and embryonic development are expressed as a function of stimulation cycle number. Neither fertilization nor blastocyst rates were affected significantly by stimulation cycle when naïve animals were contrasted to monkeys stimulated four times [71 ± 6% versus 69 ± 19% fertilization rate, P = NS (> 0.1); 41 ± 7% versus 56 ± 29% blastocyst rate, P = NS (> 0.1)]. Thus, oocyte developmental potential seems unrelated to the number of stimulation cycles.


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Table III. Effect of the number of follicular stimulation cycles on fertilization and in-vitro development of ICSI-produced embryos in the rhesus monkey
 
A potential source of variation contributing to oocyte developmental potential is the male; this has been summarized in Table IVGo as a function of oocyte/embryo performance for each of the nine sperm donors employed. Fertilization rates, when expressed as total oocytes fertilized (range 54–85%) or as replicate means (range 64–87%), were not significantly different between the nine donors. In contrast, the average blastocyst rate per replicate varied almost 4-fold, from a low of 19% to a high of 73%. Every sperm donor had at least two blastocyst-competent replicates, although the percentage of competent replicates varied from 40% to 100% among males (Table IVGo). Four donors (numbers 1–4) sired only blastocyst competent embryo cohorts, while the remaining five (numbers 5–9) had at least one incompetent cohort. Assuming that blastocyst-incompetent replicates reflect oocyte and not sperm quality, incompetent replicates were removed from analysis and blastocyst rates were re-expressed for blastocyst-competent replicates only (Table IVGo). The variation between sperm donors decreased from almost 4-fold to 1.5-fold, consistent with the conclusion that oocyte developmental competence was not highly dependent on the sperm donor.


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Table IV. The effect of the sperm donor on fertilization by ICSI and embryonic development in the rhesus monkey
 
Finally, we asked whether the individual oocyte donor determined oocyte quality. Of the 44 monkeys that contributed oocytes, 13 had at least one cycle with oocytes determined to be blastocyst-incompetent, and the expectation was—if animal-dependent oocyte quality is a major variable—that subsequent cycles would also give incompetent oocytes. However, of these 13 animals, seven were stimulated multiple times and only one animal produced blastocyst-incompetent oocytes from more than one cycle. Six of these animals, in addition to contributing incompetent oocyte cohorts, provided oocyte replicates that progressed to the blastocyst stage at a normal rate. Therefore, individual monkeys can contribute both competent and incompetent oocyte cohorts, suggesting that oocyte developmental potential is not exclusively dictated by the individual oocyte donor.

Embryo transfer
To assess in-vivo developmental potential, ICSI-produced blastocysts (Figure 5AGo) were transferred into recipient monkeys. Embryos from 11 separate experiments, ranging in stage from the compact morula to the expanded blastocyst, were transferred into 12 recipients, between days 3 and 6 after ovulation (Table VGo). Of the 12 transfers, three pregnancies were established based initially on oestradiol/progesterone profiles. Later, ultrasonography confirmed fetal heartbeats and normal fetal development. All recipients that became pregnant were at day 4 of the menstrual cycle when the transfers occurred, and all received at least one expanded, day-8 blastocyst. Two of these transfers involved transcervical replacement of the embryo, which are the first reported with blastocysts. The pregnancy rate for day 4 recipients was 60% (3/5) while day 3, 5 or 6 recipients failed to become pregnant. The birth of one female monkey (Figure 5BGo) was recorded on August 1, 1999 (at 165 days of gestation and 360 g at birth) and post-natal development has been unremarkable. The other two pregnancies are ongoing and similarly unremarkable.



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Figure 5. Rhesus monkey blastocysts (A) were transferred to recipients, with the resultant birth of a normal female rhesus monkey named `Blastulina' (B).

 

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Table V. Summary of surgical and non-surgical (transcervical) embryo transfer (ET) attempts in the rhesus monkey
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Note added in proof
 References
 
In this study with rhesus monkeys, seasonal and repeated follicular stimulation effects on ovarian responsiveness and embryonic development are described. Seasonal anovulation in the rhesus monkey has been reported (Walker et al., 1984Go; Johnsen and Whitehair, 1986Go), and was expressed here as a decrease in the number of mensing animals and the number responding to follicular stimulation. However, it can be concluded from present observations that if an animal is cycling, even in July, and responds to exogenous gonadotrophin exposure, the oocytes recovered and embryos produced are normal as defined by the ability to fertilize following ICSI and develop to the blastocyst stage in vitro. The fixed-length protocol used in the present study for follicular stimulation based on human recombinant gonadotrophins is highly efficacious, reasonably consistent, and results in an increased yield of oocytes compared with protocols based on the use of urinary gonadotrophin preparations (Lanzendorf et al., 1990bGo; Weston and Wolf, 1996Go). Such urinary preparations suffer from the tendency to elicit neutralizing antibodies inducing ovarian refractoriness after one to two cycles of stimulation (Stouffer et al., 1983Go). The ability to recover mature oocytes from rhesus monkeys after three follicular stimulation cycles using recombinant human gonadotrophins has been described (Zelinski-Wooten et al., 1996Go), and in the present study four cycles were possible in selected animals.

A retrospective assessment of oocyte competence based upon the ability of ICSI-produced embryos to grow to blastocysts has also been introduced. Oocyte evaluation of competence should be of assistance in the perfection of in-vitro manipulations, including twinning and nuclear transfer where efficiencies may be low. In these situations, it is desirable to distinguish between failed experiments due to the application of invasive technology versus those that fail because of oocyte incompetence. Despite consideration of several potential contributors to oocyte competence, it was not possible to identify a single overriding parameter that correlates with competence, and it must be concluded that in the rhesus monkey, oocyte quality is a unique characteristic of each individual stimulation cycle. Some variability in fertilization and embryonic development under in-vitro conditions is expected reflecting extrinsic differences in media, serum components, cell co-cultures and micromanipulation. Additionally, intrinsic oocyte differences are expected which may reflect discrepancies between nuclear and cytoplasmic maturation. Despite the selection of mature oocytes based on the appearance of the first polar body which presumes nuclear maturation, no convenient measure of cytoplasmic maturity was identified, apart from the ability of the oocyte to activate and support embryonic development. In the human, oocyte and embryo parameters associated with the quality of the oocyte/early embryo have been correlated to pregnancy outcome (Serhal et al., 1997Go; Xia, 1997Go; Ebner et al., 1999Go; Tesarik and Greco, 1999Go); however, similar studies in the monkey have not been addressed. It is certainly clear that cytoplasmic maturation is critical to the production of viable embryos. Previously, others (Flood et al., 1990Go) tested the hypothesis that in-vitro-matured monkey oocytes lacked essential cytoplasmic factors by conducting ooplasmic transfusions from MII to GV oocytes, rendering the latter developmentally competent.

Confounding factors that may participate in generating intrinsic oocyte variability include prior ovarian exposure to gonadotrophins, the degree of ovarian stimulation, and the age of the oocyte donor. Whether or not oocyte quality is dependent on the effectiveness of the follicular stimulation cycle, the number of repeat exposures, the dosage, or the number of follicles recruited is unknown, especially in a situation where human hormones are employed in the monkey. However, no evidence could be provided for this case in the present study. The use of fixed-length rather than individualized follicular-stimulation protocols may contribute to oocyte quality differences. The fixed stimulation protocol is convenient for advance scheduling by a busy surgical service, though it seems inevitable that immature and postmature oocyte recovery would result occasionally. Maternal age, which is a major factor in success in women undergoing assisted reproduction, would be of minor concern here because our selection based on menstrual cycle history and age should preclude perimenopausal animals. Similarly, infertility factors in these oocyte donors should be minimal as all are presumed fertile or pregnancy proven. Finally, since removal of blastocyst-incompetent experiments from sperm-donor evaluation eliminated differences in averaged monthly blastocyst development rates, it was concluded that spermatozoa from fertile males are equivalent (at least when delivered by ICSI) and do not contribute to developmental variability.

Reliable methods for the routine culture of rhesus monkey embryos to the blastocyst stage in vitro have not been rigorously tested on large numbers of embryos. The highest developmental efficiencies (66% expanded blastocyst) observed by this laboratory group involved the use of KSOM or CMRL media supplemented with serum (Weston and Wolf, 1996Go). However, subsequent efforts to replicate these results have been problematic, perhaps reflecting differences in serum lots. Therefore, it has been concluded that the use of co-cultures, despite the increased workload, is optimal at the present time. In the blastocyst-competent group, a blastocyst rate of 59% was observed, comparable with the value cited above and with that expected for women acting as oocyte donors where embryos are cultured in sequential media (Patton et al., 1999Go).

The successful birth of a normal female monkey, and two ongoing pregnancies following the transfer of ICSI-generated blastocysts, are an important landmark in our continuing efforts to produce offspring from assisted reproduction. First, the pregnancies provide evidence that our experimental system is consistent with normal fetal development in vivo. Second, the use of a transcervical approach in two of the pregnancies should facilitate future efforts to propagate monkeys. Previously, a non-surgical approach has been employed successfully with early cleavage-stage embryos in naturally cycling females (Boatman and Bavister, 1984Go). If consistency was achieved in establishing pregnancy by a transcervical approach in prescreened recipients, embryo transfers could be attempted on a monthly rather than a bimonthly basis as required with a transabdominal, surgical procedure where a rest cycle is mandatory. Furthermore, a non-surgical approach would be more amenable to field application. Finally, use of this system to culture embryos to an advanced stage should improve implantation and pregnancy rates secondary to the transfer of self-selected embryos of greater developmental potential. This argument forms the basis for the move from day 2/3 to day 5/6 transfers in women where sequential media systems have been applied successfully (Gardner and Schoolcraft, 1998Go). The low implantation efficiency seen overall in our non-surgical embryo transfer efforts to date (2/11) may reflect the technical challenge of passing a long, tortuous cervix. However, the transfer of older but slower-growing embryos into a young host uterus, i.e. day 8 expanded blastocysts into day 4 recipients, was associated with a 60% pregnancy rate and a 50% implantation rate—a rate similar to that reported by others (Ghosh et al., 1994Go). This finding suggests that early efforts at transferring embryos in synchrony with the host uterus may have been inappropriate.

In conclusion, we have demonstrated that the creation of ICSI-produced embryos with the potential to develop to the blastocyst stage on BRL cell co-culture is routine. This allows the definition of a competence screening programme that could identify blastocyst-incompetent oocyte cohorts, at least retrospectively. Since we were unable to identify a single extrinsic or intrinsic parameter that correlated with oocyte developmental competence, it was concluded that this capacity is a unique characteristic of each individual stimulation cycle. We have also demonstrated that blastocysts produced this way are viable and capable of supporting pregnancy following transfer, despite slower growth rates and higher cell numbers (Weston and Wolf, 1996Go; Ghosh et al., 2000Go). Finally, successful pregnancies following non-surgical embryo transfer should encourage further efforts to define the window of implantation and perfect this approach to propagating monkeys through the application of assisted reproduction.


    Note added in proof
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Note added in proof
 References
 
Two additional births were recorded on August 19, 2000 and August 30, 2000; a female (520 g at birth at 159 days) and a male (467 g delivered by Caesarian section at 170 days) respectively.


    Acknowledgments
 
The authors would like to recognize the contributions of Drs Dave Hess and John Fanton for hormone assay and surgical assistance respectively, and Kevin Grund, Victoria Haight and Julianne White for technical and secretarial support. The ART core facility of the ORPRC assisted by providing semen samples. This study was supported by NIH grants RR12804, A142709, PO1A136353, RR00163 and HD 18185 to D.P.W. Finally, the authors would 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.


    Notes
 
4 To whom correspondence should be addressed at: Division of Reproductive Sciences, Oregon Regional Primate Research Center, Beaverton, OR 97006-3348. E-mail: wolfd{at}ohsu.edu Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Note added in proof
 References
 
Bavister, B.D., Boatman, D.E., Collins, K. et al. (1984) Birth of rhesus monkey infant after in vitro fertilization and nonsurgical embryo transfer. Proc. Natl Acad Sci. USA, 81, 2218–2222.[Abstract]

Boatman, D.E. (1987) In vitro growth of non-human primate pre- and peri-implantation embryos. In Bavister, B.D. (ed.), The Mammalian Preimplantation Embryo: Regulation of Growth and Differentiation in vitro. Plenum Press, New York, pp. 273–308.

Boatman, D.E. and Bavister, B.D. (1984) Stimulation of rhesus monkey sperm capacitation by cyclic nucleotide mediators. J. Reprod. Fertil., 71, 357–366.[Abstract]

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Submitted on June 7, 2000; accepted on September 20, 2000.