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Regular Article |
a L'Alliance Boviteq, Inc., St-Hyacinthe, Quebec, Canada J2S 7A9
b IVF Labs, LLC, Salt Lake City, Utah 84117
c Faculté Des Sciences de l'Agriculture et de l'Alimentation, Département des Sciences Animales, Université Laval, Ste-Foy, Québec, Canada G1K 7P4
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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0.05) of in vitro culture. If a 48-h coasting period was used, LH injection did not affect the rates of blastocyst production. When donors were administered six doses of FSH with a 48-h coasting period, the highest results, although not significant (P < 0.08), were obtained when animals received LH 6 h before OPU, with 80% ± 9% (mean ± SEM) blastocysts and 0.8 ± 0.09 embryo produced per COC retrieved per heifer at Day 8 of culture. Never has in vitro technology been so close to producing 100% developmentally competent COC.
early development, follicle, in vitro fertilization, ovum, ovum pick-up/transport
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Many studies have examined different follicular characteristics in hope of discovering what factors are associated with increased developmental competence, such as follicular size [1, 5–7], follicular health [1, 7], hormonal profile [8], and ovarian status [7, 9]. Although these studies offer important clues regarding the follicular traits associated with developmental potential, it has been impossible to create ideal in vitro conditions to produce 100% competent COC. The problem with many studies is that, although they are set up to answer one question, the experimental design is confounded by two phenomena: follicular maturation versus oocyte maturation. Our laboratory has demonstrated that these two phenomena may be uncoupled [10], and this concept has also been discussed by others [11, 12]. Therefore, by altering follicular development to produce a cohort of large, healthy follicles, oocyte maturation may very well be affected, resulting in a large population of COC that appear healthy but that remain developmentally incompetent.
Our laboratory has demonstrated that administering hormones to an animal to generate a population of large, healthy follicles in the growth phase will result in a population of COC with suboptimal developmental potential [10]. This has also been demonstrated by other groups during studies in which administration of FSH did not increase the percentage of blastocysts produced when compared to nontreated animals and, in fact, actually reduced the developmental competence of the COC collected [6]. A recent publication demonstrated that multiple doses of FSH had no significant effect on the embryo production rate and only slightly increased the number of transferable embryos [12]. Clearly, simply forcing a population of follicles in the growth phase by administering FSH will not provide COC with an ideal follicular environment in which to acquire developmental competence. We have also demonstrated that the "coasting" period between hormonal stimulation and ovary collection [13] as well as the time interval between ovary collection and oocyte aspiration [14] affect significantly the developmental potential of COC. In both situations, follicles driven into phases of pseudodominance or early atresia will provide COC with an ideal environment in which to acquire developmental competence, and this phenomenon has also been demonstrated by others [7, 9]. In two studies [13, 14] we demonstrated that coasting will result in more follicles with distinct signs of follicular atresia, and that the COC will be the last to show signs of atresia. These follicular conditions closely mimic those found just before or during ovulation [15]. Clearly, COC will possess a reduced developmental potential when collected during the growth phase of the dominance period of follicular development [16, 17]. Additionally, LH pulsatility is necessary to maintain an elevated estradiol concentration and to render COC competent [8]. With the introduction of highly purified FSH preparations in many human in vitro fertilization (IVF) programs, many laboratories have examined the necessity of LH during ovarian stimulation protocols. Many papers point to the need for some LH activity during oocyte maturation to obtain developmentally competent COC, high fertilization and embryonic development rates, and increased pregnancy rates (for review, see [18] and [19]).
Bovine ovarian physiology has been, and continues to be, intensively studied for basic and applied reasons. The accumulation of physiological information during the last few years has enabled the design of very effective methods for obtaining large numbers of viable embryos following IVM. These methods usually involve the removal of all medium and large follicles via transvaginal aspiration, which creates an endogenous rise in FSH [20, 21]. Two days later, the follicular wave is sustained by multiple injections of FSH over 2–4 days. Additionally, we have demonstrated that, if follicular stimulation is diminished or ceased (i.e., FSH starvation) for either 33 or 48 h following the last FSH injection, a coasting period is created that mimics the in vivo early dominance [13]. In this study, we examined the effects of using four or six injections of FSH, different coasting periods (33 or 48 h), and injection of LH 6 h before oocyte recovery on the quality of COC collected.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Ovarian Stimulation Treatment
Experiment 1: four doses of Folltropin
Eight cyclic Holstein heifers were synchronized by inserting a progesterone-releasing device (CIDR; donated by Vetrepharm Canada, London, ON, Canada) for 9 days and injected with a prostaglandin F2
analogue (Estrumate, 2 ml i.m.; Shering Canada, Pointe-Claire, PQ, Canada) on removal of the CIDR. Superstimulation was initiated between Days 6 and 10 of the estrous cycle, and the dominant follicle was aspirated 2 days before administration of hormones. The superstimulation regimen consisted of four constant doses of FSH (Folltropin, 200 mg i.m. total; donated by Vetrepharm Canada) administered in constant doses every 12 h. In half the heifers, LH (Lutropin; Vetrepharm Canada) was administered i.v. 6 h before follicular aspiration. Only immature COC were collected, because it has been demonstrated previously that it takes 9 h after LH for COC to mature in vivo [22]. The COC were collected either 33 or 48 h following the last FSH injection (i.e., coasting period). Stimulation was done over a 4-mo period; each heifer was randomly administered a different superstimulation treatment each month. Using transvaginal ultrasonography, follicular diameters were measured, and COC were aspirated.
Experiment 2: six doses of Folltropin Six cyclic Holstein heifers were synchronized as described above. The superstimulation regimen consisted of six constant doses of FSH (Folltropin, 300 mg i.m. total) administered in constant doses every 12 h. In half the heifers, LH (Lutropin) was administered 6 h before follicular aspiration. Using transvaginal ultrasonography, follicular diameters were measured, and COC were aspirated 48 h after the last FSH injection (coasting period). Stimulation was done over a 2-mo period; each heifer was randomly administered one of the two superstimulation protocols (with or without LH) each month.
Effect of ovarian stimulation and COC retrieval The effect of repeated ovarian stimulation and COC retrieval on the number and quality of oocytes collected has been examined in bovine [23] and ovine [24] models. These studies demonstrate that repeated ovum pick-up on a weekly basis does not affect the number of follicles, quality of oocytes collected, ovarian structure, and subsequent ovarian function. In both experiments described here, animals were collected on a monthly basis, and no effect of this collection was seen on COC characteristics.
Oocyte Recovery and Classification
Follicles were visualized with a 5-MHz, transvaginal, Hitachi EUB-405 Plus Veterinary Ultrasound Transducer (Products Group International, Inc., Lyons, CO). Oocytes were recovered from follicles using the ultrasound-guided transvaginal approach. Oocytes were collected in a 50-ml conical tube containing HEPES-buffered Tyrode medium supplemented with 0.3% (w/v) bovine serum albumin (BSA fraction V), 0.2 mM pyruvic acid, 50 µg/ml of gentamicin, and 1000 IU of Hepalean (Organon Teknika, Toronto, ON, Canada). The COC were graded based on the number of cumulus layers and washed three times in Hepes-buffered Tyrode medium (TLH) medium containing 10% (v/v) fetal bovine serum (FBS), 0.2 mM pyruvic acid, and 50 µg /ml of gentamicin. Only COC with at least 2–3 layers of compact cumulus were placed in pre-equilibrated maturation medium consisting of TCM-199 with Earle salts and bicarbonate, 10% FBS, 0.5 µg/ml of FSH (Folltropin), 5 µg/ml of LH (Lutropin), 1 µg/ml of estradiol-17ß, 0.2 mM pyruvic acid, and 50 µg/ml of gentamicin.
IVM, IVF, and In Vitro Development
One to five or 6–10 COC were placed for 24 h in 25- or 50-µl droplets of maturation medium, respectively. The droplets, covered with pre-equilibrated mineral oil, were preincubated under culture conditions for at least 2 h in an incubator at 38.5°C containing an atmosphere of 5% CO2 and 95% air with 100% humidity. Mature COC were washed three times in TLH medium containing 0.3% BSA (fatty acid-free), 0.2 mM pyruvic acid, and 50 µg/ml of gentamicin. Up to five COC were then placed in 40-µl droplets of fertilization medium consisting of Tyrode lactate medium, 0.6% BSA (fatty acid-free), 0.2 mM pyruvic acid, and 50 µg/ml of gentamicin. Following transfer of the COC, heparin (final concentration, 2 µg/ml) and PHE (2 mM penicillamine, 1 mM hypotaurine, and 250 mM epinephrine) were added to each droplet. Two straws of frozen semen from the same proven bull were thawed in a 35°C water bath for 1 min and then separated by layering on a Percoll gradient as previously described [25]. To each droplet, 2 µl of sperm suspension (final concentration, 1 x 106 cells/ml) were added. After 15–18 h of fertilization, the presumptive zygotes were washed three times in TLH medium containing 10% FBS, 0.2 mM pyruvic acid, and 50 µg/ml of gentamicin. During the washings, the zygotes were denuded of their cumulus using finely stretched pipettes. The denuded zygotes were then transferred in 50-µl droplets of development medium consisting of B2 (Laboratoire C.C.D., Paris, France), 10% estrus cow serum, 0.2 mM pyruvic acid, and 50 µg/ml of gentamicin and cocultured with BRL (buffalo rat liver) cells. After 1 and 2 days of culture, fresh medium was added, and cleavage rates were recorded. After 4 days of culture, embryos were transferred to new droplets. Morula and blastocyst production rates were recorded at 4 days and at 6.5–7.5 days of culture, respectively.
Statistical Analysis
Data were arcsine transformed when the raw data failed to pass a test of normality. Percentages of blastocysts and embryo production rates on Days 7 and 8 as well as percentages of follicles between 5 and 10 mm were analyzed using a general linear models procedure [26]. The P values generated by the general models for percentages of blastocysts and embryo production rates were 0.1 (Day 7) and 0.03 (Day 8) for experiment 1 and 0.28 (Day 7) and 0.01 (Day 8) for experiment 2. The P value for percentage of follicles between 5 and 10 mm for experiment 1 was 0.015. The different variables examined within each model were heifer, coasting, LH administration, and LH x coasting (two-way interaction). Orthogonal contrasts were performed to determine significant differences between means and are presented below. Apart from Table 1, data were expressed as mean percentage (±SEM) per heifer per aspiration cycle. A P value of 0.05 or less was considered to be significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The coasting period affected significantly (P = 0.003) the distribution of follicles following four injections of FSH (Fig. 1). Significantly more follicles between 5 and 10 mm were present on the ovaries when aspiration was commenced 48 h after the last FSH injection in comparison to when aspiration was commenced at 33 h. No significant differences were apparent in the distribution of types of oocytes collected between the different treatment groups analyzed (Table 1). Because a strong tendency (P = 0.06) for the LH x coasting interaction was found for rates of blastocysts, and because it makes interpretation of the data easier, results for 33 h versus 48 h of coasting are presented separately (Fig. 2). When 33 h separated the last FSH injection and oocyte aspiration, significantly more blastocysts were produced (Fig. 2) and higher embryo production rates were achieved (Table 2) on Days 7 (P = 0.03) and 8 (P = 0.057) if LH was administered 6 h before follicular aspiration of follicles 5 mm or larger. If a 48-h coasting period was present, administration of LH did not affect the rates of blastocyst or embryo production on Days 7 and 8. Furthermore, all treatments had no effect on cleavage rates and morula production rates (Fig. 2).
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Experiment 2: Six Doses of Folltropin
When heifers were injected with six doses of FSH and oocytes retrieved 48 h after the last injection, administration of LH 6 h before follicular aspiration of follicles 5 mm or larger had a tendency (P < 0.08) to increase the percentage of Day 8 blastocysts produced (Fig. 3) and the embryo production rate (Table 2). Overall, six doses of FSH plus LH resulted in the highest rates of blastocysts, which were as high as 80.4% ± 9.4%, and in 0.8 ± 0.09 embryo produced per COC retrieved per heifer on Day 8. Administration of LH did not affect the distribution of follicles or the types of oocytes collected (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Treatment with FSH will increase the numbers of medium- and large-sized follicles [12, 27, 28]. When administering four doses of FSH, a longer "coasting" period (48 h vs. 33 h) clearly resulted in a greater percentage of 5- to 10-mm follicles. Although FSH support was discontinued for an additional 15 h when a 48-h coasting period was used, follicular growth was not inhibited, because a reduction in the percentage of smaller follicles and an increase of medium-sized follicles was observed. Since others have demonstrated that COC from larger follicles are developmentally more competent [1, 5–7], it seems reasonable to include a 48-h coasting period when this superstimulation regimen is used. Although the coasting period and LH administration did not affect the types of COC collected, a significant effect on the percentage of blastocysts and embryo production rates was found. With four injections of FSH plus 33 h of coasting, administering LH increased the percentage of blastocysts and the embryo production rates on Days 7 and 8. It seems plausible that, following four injections of FSH, follicles are still in the growing phase following 33 h of coasting, and that this environment is not ideal for COC to acquire full developmental competence [10]. Administering LH 6 h before ovum pick-up may have induced follicular changes much like those seen during ovulation that permit COC to complete cytoplasmic maturation [13, 14]. This agrees with the concept that superovulation can result in an uncoupling of follicular and oocyte maturation [10–12]. Modifications to superstimulation protocols, such as including a coasting period and LH administration, permit COC to complete cytoplasmic maturation and to become developmentally competent. Furthermore, only immature COC were collected in the present study, because they were aspirated 6 h after LH and, as demonstrated previously, it takes 9 h after LH for COC to mature in vivo [22].
It has been proposed that, during the dominance phase of an unstimulated cycle, the dominant follicle will suppress acquisition of developmental competence by its own COC and by those from subordinate follicles [16, 17]. Loss of "functional" dominance will signal the COC and may increase their intrinsic developmental competence. Ovulation offers COC the opportunity to escape the atretic fate of the follicles [7]. Administration of LH with 33 h of coasting could very well mimic these conditions, creating an ideal follicular environment in which to complete cytoplasmic maturation. Slightly prolonging the follicular phase and inducing follicular atresia does not reduce the developmental potential of the COC collected [29]. Furthermore, we have demonstrated that a reduced rate of follicular growth will induce the COC to acquire developmental competence and will increase the rates of in vitro embryo production [13, 14]. Therefore, by ceasing FSH support for 48 h to induce a progressive arrest in follicular growth, COC were induced to acquire developmental competence in vivo, a phenomenon previously demonstrated in our laboratory [30]. In this case, administering LH did not provide any significant additional stimulus to increase the percentage of competent COC. On the other hand, with only 33 h of coasting, follicles possibly have not reached a pseudoplateau or atretic phase as advanced as those follicles subjected to 48 h of coasting, and administering LH induced COC to differentiate rapidly. We have seen a similar phenomenon in which COC were left in postmortem ovaries for 4 h, which induced them to acquire higher developmental competence and to produce more blastocysts [14]. Growing evidence indicates an important role for both FSH and LH in folliculogenesis and oocyte maturation. In human IVF, a minimal level of LH activity during exogenous stimulation protocols is desired to optimize ovulation induction in patients [18]. In fact, it has been postulated that profound LH suppression could affect optimal oocyte maturation and/or endometrial development [19]. Including LH in ovarian stimulation protocols using highly purified FSH preparations will affect blastocyst quality, resulting in an increased embryo implantation and pregnancy rate [31].
The best results were obtained when animals received six injections of FSH with a 48-h coasting period. Administering LH 6 h before ovum pick-up resulted in 100% cleavage and 81.9% ± 8.7% morula. Furthermore, virtually every morula proceeded to the blastocyst stage after 8 days of culture, resulting in 80.4% ± 9.4% blastocysts, which tended to be more blastocysts than found in the superstimulation protocol without LH. The ultimate goal for every reproductive physiologist is to obtain an embryo production rate (mean number of embryos produced per COC inseminated per heifer) of one, but until today, results have been quite variable. Embryo production rates have rarely surpassed 0.4 [12, 26, 32, 33]. With these results, we obtained an embryo production rate of 0.8 ± 0.09 on Day 8 of culture. Never have we been so close to obtaining 100% developmentally competent COC to produce viable, transferable blastocysts. Although these animals were housed within a commercial setting where many external parameters were controlled [4], extrinsic factors, such as the animal's genetic merit and body condition score [34], can affect the quality of the COC produced and the resulting rates of embryo production.
In vivo, a dominant follicle will survive many days with little FSH support and utilize endogenous LH to complete follicular maturation, culminating in the ovulation of a developmentally competent oocyte [35]. The kinetics of the superstimulation protocols described here involved production of many medium- to large-sized (i.e., "dominant-type") follicles, starvation of these follicles by ceasing FSH support, and completion of both follicular and oocyte maturation by administering LH before ovum pick-up. This novel approach should be further investigated to get a complete understanding of this phenomenon and to eventually apply this physiological evidence to other mammalian species. In human IVF, superovulation protocols still consist of supporting follicular growth with gonadotropins until hCG is administered before ovum pick-up, and application of these new ideas, either in part or in full, could lead to new clinical approaches [36, 37].
Overall, these results confirm our hypothesis that inducing follicular growth to simply increase the number of medium- and large-sized follicles is not sufficient to produce maximal numbers of competent COC. Induction of a preovulatory-type follicular environment is necessary to trigger COC to complete their cytoplasmic maturation so that oocytes are developmentally competent even before maturing them in vitro. This suggests that inducing follicular maturation does not necessarily result in oocyte competence, and that both phenomena must be achieved to produce a maximal number of developmentally competent COC. A 48-h coasting period plus LH administration, in association with a standard FSH superstimulation protocol, is sufficient to create these optimal in vivo follicular conditions. The embryo transfer center in which these experiments were conducted has since adopted these protocols and produced many live births following transfer of such embryos. Therefore, we are convinced that the quality of the embryos produced with these protocols is of comparable, if not better, quality compared to embryos produced using standard stimulation protocols, but a controlled comparison will be necessary to prove this concept.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by NRC (IRAP), SEMEX Canada, NSERC, and Pacific Fertility Medical Centers. 
2 Correspondence. FAX: 418 656 3766; marc-andre.sirard{at}crbr.ulaval.ca
Accepted: August 13, 2001.
Received: February 28, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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5 mm after 7 or 8 days of in vitro development.a