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Reproductive Technology |
Department of Physiology and Pharmacology3
Department of Pathology,4 Wake Forest University Health Sciences, Winston-Salem, North Carolina 27157
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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assisted reproductive technology, embryo, follicular development, in vitro fertilization, oocyte development
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The HPG axes of rhesus and cynomolgus macaques display marked similarities to that of humans. Gonadotropin secretion is ultimately controlled by ovarian steroid feedback to the pituitary and hypothalamus [1]. A preovulatory GnRH and resulting LH/FSH surge are necessary for initiation of final oocyte maturation, ovulation, and progesterone synthesis. Menstrual cycle length, reproductive endocrine status, and the release of only one or two mature oocytes are similar as well [2–5]. Fertilization events and embryo development are also strikingly similar and can be reproduced in vitro [6–9].
Control mechanisms for reproductive success in both human and nonhuman primates rely on the interactions of hypothalamic GnRH, pituitary LH/FSH, and ovarian steroids. The triad of hypothalamic down regulation of GnRH, exogenous gonadotropin administration, and timed initiation of ovulation increases mature oocyte yield in both species by 10-fold, and this multifaceted approach has been used in human in vitro fertilization clinics for more than 30 years. The goal for ART and researchers has been to increase the number of mature oocytes available for manipulation. For ART clinics, the ultimate goal is to enhance reproductive outcome. The aims for basic research include the opportunity to better understand nonhuman primate reproductive physiology and to extend the acquired knowledge to both human and nonhuman applications. In light of ethics, subject availability, and legal constraints, advances in these fields have primarily been developed in macaques because of their similarities in reproductive function and control.
Four general categories of reproductive-cycle stimulations are reported in human and nonhuman primates: gonadotropin-administration only; gonadotropin administration followed by hCG for ovulation induction without down regulation; short-acting GnRH agonist down regulation, gonadotropins, and hCG; and finally, short-acting GnRH antagonist down regulation, gonadotropins, and hCG.
The first and second categories consist of the administration of gonadotropins with or without hCG. Early research and some current studies have used this approach for oocyte procurement [10–15]. Gonadotropin administration alone limits initiation of stimulation to the first 3 days of menses and depends on spontaneous ovulation for final oocyte maturation and release. The addition of hCG to gonadotropin cycles allows for predicted timing of ovulation. Clinically, premature or spontaneous ovulation occurs in approximately 20% of these cycles [16].
The third category consists of short-acting GnRH agonist down regulation, gonadotropin administration, and hCG, which is the methodology primarily used in ART facilities for in vitro fertilization (IVF) procedures. The GnRH agonists act by causing down regulation and desensitization of anterior pituitary gonadotropin receptors. An initial stimulatory effect results in the immediate release of LH and FSH. One to two weeks of agonist down regulation are necessary for the onset of the inhibitory phase [17]. Chronic administration allows exquisite fine-tuning of gonadotropin administration, regulation of follicle development, timed oocyte release, and maximum yield of mature oocytes. Cycle starts can be easily manipulated for scheduling purposes. The incidence of premature ovulation drops to 2% [16].
For many years, the gold standard for down regulation in IVF cycles has consisted of using a short-acting GnRH agonist, such as Lupron (Tap Pharmaceuticals, Inc., Lake Forest, IL). Lupron is administered s.c. once daily beginning on Cycle Day 21–23 of the luteal phase of the previous cycle and is continued through the stimulation phase of the second cycle until hCG administration occurs. This approach requires daily injections during the down-regulation phase and then multiple daily injections (Lupron and gonadotropins) during the stimulation phase. Short-acting agonist use in nonhuman primates (squirrel monkeys) has been reported [18].
The fourth category uses the coadministration of a short-acting GnRH antagonist, such as Antide (Ares-Serono, Geneva, Switzerland) or Antagon (Organon, Inc., West Orange, NJ) and gonadotropins followed by hCG. Hypothalamic control is disrupted by rapid interference with endogenous GnRH activity [19]. This immediate action prevents premature ovulation and lacks the initial stimulatory phenomenon seen with agonist use [20]. This method first appeared in the nonhuman primate literature [21–24], but during the past two years, it has acquired approval from the U.S. Food and Drug Administration and is now being used clinically [25, 26]. This approach decreases the interval of down regulation, because the antagonist is only used for 5–6 days of the cycle, during the latter part of the stimulation period.
A potential alternative for down regulation in COH cycles is the use of a long-acting GnRH agonist. Indications for use of the long-acting GnRH-agonist Lupron Depot (3.75 mg, 1-mo form) include treatment of endometriosis, pelvic pain, and uterine fibroids. Hsieh [27] reported in a retrospective clinical study that a single (1.88 mg) dose administered to women during the luteal phase of the previous cycle exhibited equivalent down regulation with similar numbers of oocytes, embryos, and pregnancy rates after COH and IVF. As a drug indication, this method "may be an appropriate alternative to daily SQ [s.c.] doses of short-acting Lupron for pituitary suppression in ovarian stimulation for in-vitro fertilization" [17]. To date, the use of a long-acting GnRH agonist for nonhuman primate COH/IVF has been limited to baboons [28].
The goals of the present study were to develop a combined down-regulation/COH protocol that maximized our female cynomolgus resource pool. Specifically, we aimed to increase the number of animals at the optimal cycle phase for stimulation starts and to consistently produce higher numbers of mature oocytes from these stimulations. Oocyte quality was assessed by the ability to undergo fertilization or parthenogenic activation and subsequent cleavage.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Ten adult (mean age, 8.7 years) female cynomolgus monkeys (Macaca fascicularis) were individually housed in stainless-steel primate cages (67 x 35 x 79 inches; Allentown Caging Equipment Co., Inc., Allentown, NJ). Cages were provided with divider panels, allowing monkeys to be horizontally pair-housed when possible. Humidity and temperature (72–74°F) were controlled, and a 12L:12D photoperiod was imposed with lights-on at 0600 h. Monkeys were maintained on Lab Diet 5045 High Protein Monkey Diet (PMI Nutrition International, Inc., Bentwood, MO). The care and treatment of the monkeys were in accordance with state and federal guidelines, specifically outlined in Principles of Animal Care (NIH publication no. 85-23, revised 1995), and the procedures were reviewed and approved by the Wake Forest University Animal Care and Use Committee.
Menstrual cycle phase was determined by daily vaginal swabs and recorded to maintain accurate track of cycle status. In brief, animals were trained to approach the front of the cage and present their hindquarters. A cotton swab was gently inserted into the vaginal area, and menses was determined by visual inspection [29].
Short-Acting GnRH Agonist Down Regulation (Lupron)
Normally cycling females underwent down regulation on Cycle Day 21, 22, or 23 with daily 0.25 mg/kg s.c. injections of Lupron. The starting dose of Follistim (rhFSH; Organon) was customized for the age of the animal. Females younger than 6 yr were administered 30 IU of Follistim daily for 10 days beginning on Days 1, 2, or 3 of the following menses in addition to the morning Lupron. Females older than 6 yr were administered 30 IU of Follistim twice daily for 10 days in addition to the morning Lupron. On Day 6 of stimulation, the animals were anesthetized with 15–20 mg/kg of ketamine for follicular ultrasound, and blood was drawn for serum estradiol. Ovulation was induced by administering 2000 IU of hCG on Day 10 in addition to Lupron, and on Day 11, the animals underwent ultrasound for follicular development before oocyte retrieval.
Long-Acting GnRH Agonist Down Regulation (Lupron Depot)
Normally cycling females underwent down regulation with a single i.m. injection of 0.93 mg of Lupron Depot during the third week of the menstrual cycle. The recommended Lupron Depot dose for 1 mo of suppression for the treatment of endometriosis (3.75 mg/mo i.m.) was adjusted to 0.93 mg/mo for each animal, or one quarter of the human dose for GnRH down regulation. The starting dose of Follistim was customized in the same manner for the age of the animal and by individual retrieval data from previous cycles. On Day 6, the animals were anesthetized with ketamine for ultrasound, and blood was drawn for serum estradiol levels. On Day 10, 2000 IU of hCG were administered, and oocyte retrieval was performed 24 h later.
Hormone Analysis
Serum estradiols were performed using the DPC Immulite automated immunoassay system (Diagnostic Products Corporation, Los Angeles, CA). The LKE21 estradiol kit identifies 17ß-estradiol in a competitive immunoassay. The analytical sensitivity is 15 pg/ml, and intra- and interassay precisions were 15%–16% at low levels (46–56 pg/ml) and 6.3%–6.4% at high levels (480 pg/ml).
Sperm Collection/Preparation
Fresh sperm was collected on the day of oocyte retrieval using electroejaculation [30]. Ejaculates were collected into human tubal fluid (HTF) containing 10% human serum albumin (HSA; Fertility Technology Resources, Inc., Marietta, GA) and then centrifuged at 1200 rpm for 10 min. The supernatant was removed, and the pellet was overlaid with 1 ml of fresh HTF-10% HSA, placed in the incubator, and allowed to swim up for at least 1 h before use.
Oocyte Retrieval
Animals were sedated with ketamine (15–20 mg/kg), and transabdominal ultrasound was performed to verify the presence of follicles that were at least 4 mm in diameter (Sonosite, Bothwell, WA). If follicles were not visualized, the retrieval was cancelled. The animals were intubated and maintained on a mixture of 2% isofluorane/oxygen anesthesia, and pulse oxygen saturation and body temperature were monitored throughout the procedure. Ovaries were exposed via laparotomy. A 25-g butterfly attached to a 10-ml Air-Tite syringe (Air-Tite Products Co., Inc., Virginia Beach, VA) containing 2 ml of HTF-Hepes (Fertility Technology Resources) supplemented with 5 IU/ml of heparin (Upjohn, Kalamazoo, MI) was used for aspiration. The needle was introduced into each visible follicle while maintaining steady vacuum with the syringe. When all the follicles from one ovary were aspirated, the fluid in the syringe and tubing was flushed into a 75- x 100-mm test tube, and the second ovary was treated in the same manner.
Oocyte Isolation and Identification
Oocyte-cumulus complexes were identified and isolated using a Nikon (Southern Micro Instruments, Marietta, GA) stereo dissecting scope located in a laminar flow hood. Strips of cumulus cells devoid of oocytes were removed and placed into microdrops of HTF-10% HSA to be used for coculture with immature eggs. Oocytes were denuded of cumulus cells using hyaluronidase (80 IU/ml; Medicult, Jyllinge, Denmark). Denuded oocytes were transferred to microdrops of HTF-10% HSA overlaid with oil (Conception Technologies, San Diego, CA). The oocytes were evaluated for maturity using an Olympus IX70 inverted microscope (Olympus Corporation, Melville, NY) and then segregated for manipulation or further in vitro maturation. Mature MII oocytes capable of fertilization or activation were identified by the presence of the extruded polar body and placed into microdrops of HTF-10% HSA. Immature metaphase I and germinal vesicle-stage oocytes were placed in droplets of HTF-10% HSA containing strips of autologous cumulus cells for in vitro maturation.
Oocyte Fertilization
For intracytoplasmic sperm injection, MII oocytes were allowed to culture for 3–8 h postretrieval before insemination. Briefly, a 30-µl droplet of HTF-Hepes-10% HSA was placed in the center of the lid of a Nunc 35-mm dish (Fisher Scientific, Atlanta, GA) along with a small volume (10 µl) of prepared sperm and an equal volume of polyvinylpyrrolidone (PVP; Medicult, Jyllinge, Denmark). A 2-ml oil overlay was added so that all three microdrops were covered. Oocytes to be injected were placed in the HTF-Hepes-HSA droplet. Morphologically normal sperm were aspirated from the sperm droplet under 40x magnification and deposited in the PVP. A single sperm was then aspirated into the ICSI pipette. The oocyte was maneuvered and held so that the polar body was in either the 11-o'clock or the 7-o'clock position. The tip of the ICSI pipette was gently introduced through the zona and oolemma to the center of the oocyte. Gentle aspiration of ooplasm was performed twice, and the ooplasm and single sperm were deposited within the oocyte. Injected oocytes were returned to microdrops of HTF-10% HSA until fertilization was assessed the following morning. All manipulations were done in microdrops under oil at 37°C unless stated otherwise.
Oocyte Activation
Oocytes for parthenogenic activation were denuded of cumulus cells as described above, placed in HTF-10% HSA, and allowed to culture for 24 h. Activation was induced with modifications of a protocol used by Cibelli [31]. The MII oocytes were incubated in 5 µg/ml of cytochalasin B (Sigma, St. Louis, MO) for 30 min, followed by 5 µM ionomycin (Sigma) for 8 min and then 2 mM 4-dimethylaminopyridine (Sigma) for 3 h. Oocytes were then moved to microdrops of HTF-10% HSA for overnight culture.
Embryo Culture
Oocytes with two visible pronuclei and two polar bodies (2pn/2pb; ICSI) at 18–22 h after retrieval were isolated and placed into culture dishes containing QOne-15% HSA (Fertility Technology Resources). Oocytes that displayed one pronucleus and two polar bodies (1pn/2pb) or two pronuclei and one polar body (2pn/1pb) at 18–22 h after activation were isolated and placed in culture dishes containing QOne-15% HSA. All embryos remained in QOne until Day 3 of culture. Embryos progressing to the 8-cell stage by Day 3 were placed in microdrops of QThree-15% HSA (Fertility Technology Resources) for subsequent development. All culture was done in microdrops under oil at 37°C and 5%CO2 unless stated otherwise.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Five cycles were initiated using short-acting Lupron and Follistim. Day 6 ultrasound revealed cystic ovaries in one animal, resulting in a cancelled cycle. Follicular development was observed in three animals. Day 6 serum estradiol levels ranged from 852 to 7150pg/ml. Three of the females continued their Follistim dose, and the dose for the fourth animal was decreased because of elevated serum estradiol (7150 pg/ml). Contradictory ultrasound and estradiol results in these animals resulted in a second blood draw and ultrasound on Day 8 of stimulation. All animals had follicles present on ultrasound. Stimulation for two animals was discontinued because of extremely elevated serum estradiol (18,161 and 16,555 pg/ml). On Day 11, the two remaining animals underwent ultrasound for follicular development. Development was observed, and the animals proceeded to oocyte retrieval. A total of 14 oocytes were retrieved, for an average of 7 oocytes per retrieval. The percentage maturity rate for these retrievals was 50% after 24 h of culture (Table 1). The cancellation rate for this group was 60%. Mature oocyte numbers were insufficient to evaluate oocyte competency. The combination of down regulation and stimulation used in this series exhibited low efficiency, as reflected by the expected conventional IVF outcome and reflected an inefficient use of our nonhuman primate resources.
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Long-Acting GnRH Agonist Down Regulation
Fourteen cycles were initiated using Lupron Depot and Follistim, with 11 cycles going to retrieval based on Day 6 estradiol levels (120–1245 pg/ml) and the presence or absence of follicular development as determined by ultrasound. Cumulative estradiol data indicated that Day 6 estradiol levels between 800–1200 pg/ml resulted in optimal recovery of mature MII oocytes. Serum estradiol levels of 400 pg/ml or less on Day 6 and/or the absence of follicles on ultrasound resulted in a cancelled cycle, as did Day 6 estradiol levels greater than 2000 pg/ml. The average number of oocytes retrieved was 15.5 oocytes per retrieval, with 56% being mature after 24 h of culture (Table 1). Two of the three cancellations in this series resulted from poor response in the same animal (Day 6 estradiol levels of 120 and 250 pg/ml). The third cancellation resulted from a Day 6 estradiol level of 178 pg/ml. The cancellation rate for this group was 22%.
Oocyte Competence
Metaphase II oocytes from the last five Lupron Depot down-regulated cycles were subjected to either ICSI insemination or parthenogenic activation (Table 2 and Fig. 1). ICSI insemination resulted in a fertilization rate of 59%. Of the oocytes fertilized, 91% reached appropriate cleavage stages by Day 2 of culture, with 17% reaching the morula stage by Day 5 of culture. Parthenogenic activation of MII oocytes resulted in a 70% activation rate, with 86% reaching appropriate cleavage stages by Day 2 of culture and 29% reaching the morula stage by Day 5 of culture.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The second method of down regulation that we investigated consisted of the long-acting GnRH agonist Lupron Depot combined with COH and hCG. The long-acting agonist could be administered at any point during the third week of the menstrual cycle, as opposed to Days 21, 22, or 23 with the short-acting form. Once menses had occurred, stimulations for this group could be initiated at any point during the month following the single injection. This regimen increased the number of animals available at the appropriate phase of the menstrual cycle for group stimulations and oocyte retrievals. Using the long-acting regimen, the average number of oocytes per retrieval increased, with a slight increase in maturity rate (Table 1), and the cancellation rate for this protocol was reduced compared to the short-acting agonist protocol. We hypothesized, based on reports by Hseih et al. [27] and Cseh et al. [28] that long-acting down regulation combined with COH might be a more efficient method for oocyte production in cynomolgus macaques. Ultrasound and estradiol levels evaluated cycle progression on Day 6, and cycles were adjusted accordingly. As with the first group, the starting dose of Follistim was customized for the age of the animal. In addition, individual retrieval data from previous cycles were evaluated to assist in determining Follistim dose.
Using this protocol we observed a doubling in the number of oocytes per retrieval with a slight increase in maturity rate after 24 h of culture (Table 1). The average number of oocytes retrieved and the percentage of mature oocytes in the present study are comparable to those reported in the literature, despite differences in the nonhuman primate model, drug regimens, and media used. For example, an FSH/LH/hCG regimen without down regulation in rhesus monkeys resulted in 18 oocytes per retrieval with 63% maturity [12]. In a study examining effects of media composition on oocyte competency, a stimulation protocol used by Zheng et al. [13] consisted of FSH alone, resulting in 18.2 oocytes per retrieval and a percentage maturity across the media studied of approximately 60% in rhesus monkeys. Short-acting antagonist down regulation in rhesus monkeys has been reported by Zelinski-Wooten et al. [21], with 51% mature oocyte production, as well as by Sanchez-Partida et al. [23], with a 57% maturity rate. In addition, Wolf et al. [22] reported an average yield of 16.7 oocytes per retrieval, and Hewitson et al. [33] reported an average oocyte yield of 27 oocytes per retrieval and 53.7% maturity using short-acting antagonist down regulation. Cseh et al. [28] induced down regulation in baboons with a long-acting agonist similar to Lupron Depot (goserelin acetate) followed by a stimulation protocol. This regimen resulted in an average of 17 oocytes per retrieval. Therefore, the efficiency of a long-acting agonist for hypothalamic down regulation in the present study is reflected not only in the comparable numbers of oocyte yield and maturational status but also by the low cancellation rate. Higher numbers of cycles going to retrieval result in higher oocyte yield for research purposes and a more efficient use of our cynomolgus resource pool. These data suggest that the use of a single long-acting agonist in conjunction with COH is an efficient alternative for oocyte production in cynomolgus macaques.
Oocyte competence was evaluated by subjecting MII oocytes from the last five Lupron Depot down-regulated cycles to ICSI insemination or parthenogenic activation. The fertilization rate for ICSI was 59%, with 91% cleaving within 24 h and 17% reaching the morula stage by Day 5 of culture (Table 2). These results are similar to those obtained by Ogonuki et al. [34] in cynomolgus macaques, with a 61% ICSI fertilization rate and with 7% of oocytes fertilized proceeding to the morula stage. Again, despite differences in species and study design, our results are comparable to those reported in the literature. In rhesus monkeys, Mitalipov et al. [24] achieved a 71% ICSI fertilization rate and an 87% cleavage rate in oocytes retrieved from short-acting antagonist/FSH/LH/hCG cycles in rhesus. Wolf et al. [12] observed a 26–75% fertilization rate using conventional IVF with chemically activated sperm. Conventional IVF with chemically activated sperm in one study by Zheng et al. [13] resulted in fertilization rates in the 80% range across media groups. Interestingly, a study by Zheng et al. [14] using similar stimulation parameters reported a 54% morula rate. In an FSH-only protocol, Si et al. [15] reported an 82% conventional fertilization rate using chemically activated sperm in rhesus monkeys, with 63% reaching the morula stage. In clinical IVF cycles, a fertilization rate of at least 50% is expected, and rates of 80% or higher are usually achieved regardless of fertilization techniques. In clinical practice, successful fertilization using ICSI requires mechanical sperm membrane disruption achieved by rolling the sperm tail on the bottom of the dish with the ICSI pipette. Hewitson et al. [33] examined the ICSI procedure in rhesus monkeys and reported that sperm immobilization (membrane disruption) or additional chemical stimulation was unnecessary for the initiation of fertilization and subsequent embryo development. A 76.6% ICSI fertilization rate was reported for their study. The ICSI protocol for this study used a swim-up procedure without subsequent chemical activation for sperm isolation, and sperm were injected without sperm membrane disruption, which could account for the differences observed in fertilization rates between species. Our results indicate that the use of long-acting Lupron Depot does not have a negative impact on oocyte competency as defined by their ability to undergo fertilization and to complete cleavage stages at appropriate time intervals. The morulation rate in the present study is lower than those in some reports. This difference is more likely attributable to differences in media composition as opposed to the method of down regulation, because some of the studies cited did not use down regulation and others used an antagonist, as opposed to an agonist, for down regulation.
Mature oocytes subjected to parthenogenic activation in the present study resulted in a 70% activation rate (number of MIIs parthenogenically activated divided by the total number of MIIs going through the activation protocol), with 86% cleaving within 24 h and 29% reaching the morula stage by Day 5. Mitalipov et al. [24] used a similar parthenogenic activation protocol in rhesus monkeys, resulting in a 95% cleavage rate. Cibelli et al. [31] reported that 4 of 28 mature oocytes that were parthenogenically activated reached the blastocyst stage in cynomolgus macaques. These data also suggest that the use of a long-acting agonist for down regulation does not negatively impact oocyte competence under the conditions studied.
In summary, a single injection of long-acting Lupron Depot provided for an efficient, repeatable production of large numbers of mature MII oocytes. The window for initiating down regulation was broadened, which allowed for increased recruitment for cycle stimulations. Daily injections of the short-acting agonist could be replaced by a single injection of the long-acting form. Controlled ovarian hyperstimulation, ultrasounds, blood draws, and oocyte retrievals could be manipulated for optimal synchronization of investigators as well as veterinary and animal support staff. Cycle cancellations were minimal, which suggests that this regimen of down regulation and COH was effective for the majority of the animals in the resource pool. Oocytes retrieved from these cycles underwent fertilization or parthenogenic activation and reached cleavage stages at appropriate time intervals. The results of the present study suggest that a long-acting GnRH agonist can be an efficient alternative for GnRH down regulation when used with COH for oocyte and embryo production in cynomolgus macaques. Oocytes produced by this regimen are competent, as indicated by their ability to fertilize and cleave. Studies are currently in progress to evaluate the ability of these embryos to create live births, which is the ultimate test of an assisted reproduction stimulation protocol.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence. FAX: 336 713 7168; kagrant{at}wfubmc.edu
Received: 16 October 2002.
First decision: 9 November 2002.
Accepted: 21 January 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-hydroxy-5-pregnan-20-one in relation to menstrual cycle phase in cynomolgus monkeys (Macaca fascicularis). Psychopharmacology (Berl) 1997 130:59-68[CrossRef][Medline]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
