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
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Abstract |
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Key words: blastocyst/embryo transfer/ICSI/monkey/oocyte quality
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Introduction |
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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., 1970), and significant differences in blastocyst quality have been defined clinically (Gardner and Schoolcraft, 1998
). On the other hand, attainment of the blastocyst stage is a significant accomplishment, as the embryoif normal and diploidhas 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 822% (Boatman, 1987; Zhang et al., 1994
) to a high of 66% (Weston and Wolf, 1996
). 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., 1989
), and that pregnancy establishment may be impacted by embryouterine 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, 1984), which may act via protein kinase (Osheroff et al., 1999
). 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., 1992
) 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., 1999
). In the rhesus monkey, the participation of the sperm head in ICSI-induced oocyte activation has been documented (Meng and Wolf, 1997
) and, recently, the first ICSI monkey was born following transfer of an 8-cell stage embryo (Hewitson et al., 1999
).
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.
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Materials and methods |
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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, 1997) within 45 h after surgical retrieval employing spermatozoa collected by penile electrostimulation from pregnancy-proven males (n = 9) (Lanzendorf et al., 1990a
). 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 1620 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., 1990). 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., 1981
). 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., 1990
). 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.
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Results |
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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 4) 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 4
). 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 II
). 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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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 I), although multiple stimulations were associated with an increase in the non-response rate (Figure 1
). In Table III
, 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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Embryo transfer
To assess in-vivo developmental potential, ICSI-produced blastocysts (Figure 5A) 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 V
). 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 5B
) 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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Discussion |
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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., 1997; Xia, 1997
; Ebner et al., 1999
; Tesarik and Greco, 1999
); 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., 1990
) 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, 1996). 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., 1999
).
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, 1984). 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, 1998
). 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 ratea rate similar to that reported by others (Ghosh et al., 1994
). 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, 1996; Ghosh et al., 2000
). 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.
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Note added in proof |
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Acknowledgments |
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Notes |
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References |
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Submitted on June 7, 2000; accepted on September 20, 2000.