Prediction of ovarian response to exogenous gonadotropin stimulation: Utilization for collection of primate oocytes for fertilization in vitro

PREDICTION OF OVARIAN RESPONSE TO EXOGENOUS GONADOTROPIN STIMULATION: UTILIZATION FOR COLLECTION OF PRIMATE OOCYTES FOR FERTILIZATION IN VITRO S.E. Lanzendorf’“,

K. Gordon’, J. Toner’, G.D. Hodgen’

M.C. Mahony’,

P. Kolm’

The Jones Institute for Reproductive Medicine ‘Department of Obstetrics and Gynecology ‘Department of Research and Academic Planning Eastern Virginia Medical School Norfolk, VA 23507 Received for publication: Accepted:

,~u/uzy14, 1994 May 16, 1995

ABSTRACT The predictive value of 2 tests for ovarian response to controlled ovarian hyperstimulation in the cynomolgus monkey model was evaluated. The tests utilized were: 1) the cycle Day 3 (Day 1 = onset of menses) FSH value and 2) the acute estradiol (E,) response to a GnRH agonists (GnRHa) administered on Day 3. Both tests were performed during the cycle preceding control ovarian hyperstimulation. Subsequently, monkeys (n = 26) were stimulated with MetrodinT (Days 2-6, 25llJ/d) and PergonalT (Day 7 to hCG administration, 25lU/d). Laparoscopic oocyte retrieval was performed 32 to 34 after hCG administration. Analysis of the data revealed that Day 3 FSH values could not predict whether an animal would respond well to control ovarian hyperstimulation in a subsequent cycle (P = 0.77). However, the E, change 24 h post-GnRHa administration was significantly greater for animals responding well to control ovarian hyperstimulation compared with the animals deleted after 6 d of stimulation (P = 0.042). The mean change in E, levels in animals taken to aspiration This was 97.8 pg/ml compared with only 21.6 pg/ml for the deleted animals. differential response of E, production after GnRHa treatment was used to correctly identify (by discriminant analysis) 78% of the animals subsequently deleted for poor response. Thus, the increase in serum E, level after GnRHa, but not the basal FSH level, was found to be predictive of ovarian response to stimulation in the cynomolgus monkey. Key words:

in vitro fertilization,

control ovarian hyperstimulation

Acknowledgments The authors thank the Thomas F. and Kate Miller Jeffress Memorial Trust and The Mellon Foundation for their support of this research. We also thank Todd Goodrich and Susan Whitelock for their technical assistance, and Dara WillettLeary for editorial assistance and manuscript preparation. Dr. Susan Lanzendorf, The Jones “Correspondence and reprint requests: Institute for Reproductive Medicine, Department of Obstetrics and Gynecology, Eastern Virginia Medical School, 601 Colley Ave., Norfolk, VA 23507. Theriogenology 44:641-648, 1995 0 1995 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NV 10010

0093-691X/95/$1 0.00 SSDI OC93-691X(95)00244-8

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Theriogenology INTRODUCTION

In vitro fertilization W/F) has been performed successfully in numerous primate models, including squirrel monkeys (51, cynomolgus monkeys (I 1, rhesus monkeys (91, chimpanzees (4) and marmosets (IO). The use of these models provides valuable information not only for human reproductive technology and contraceptive research, but also for the preservation of endangered primate species and for the propagation of animals for disease models for medical research. In vitro fertilization routinely uses hormonal stimulation of the female to increase the number of growth of ovarian follicles. Due to the unavailability of pure monkey gonadotropins, human gonadotropins are used to induce controlled ovarian hyperstimlation. Unfortunately, these are allergenic to monkeys thus limiting their use to a single stimulation cycle. In addition, female monkeys, like women, manifest variable responses to control ovarian hyperstimulation with poor, good and high responses. One might also assume that, as in the human, ovarian response to stimulation may decline with chronological age. However, in many instances, investigators do not know the. actual age of the animals available to them. Considerable saving in time and resources could be achieved if monkeys that respond well to control ovarian hyperstimulation could be pre-selected. In the human, the serum FSH level on menstrual cycle Day 3 (Day 3 FSH assay) has been shown to predict outcome of control ovarian hyperstimulatian (3,11,18). In a study evaluating 1,478 IVF cases, Toner et al (18) demonstrated that basal FSH was more predictive than age for cancellation risk, peak E,, number of oocytes retrieved, and other IVF outcomes. An assay that measures ovarian function, referred to as the GnRH-a stimulation test, has also been evaluated in the human (13, 19). This assay was shown to predict ovarian response to stimulation and to provide information on the number of embryos available for transfer and cryopreservation (19).

The present study was undertaken to identify a method for predicting ovarian function in the cynomolgus monkey model. Both the Day 3 FSH assay and the GnRHa stimulation test were evaluated hyperstimulation.

for their ability to predict ovarian response to control

MATERIALS

AND METHODS

Animals Adult female cynomolgus monkeys (Macaca fascularis; n = 26) having regular menstrual cycles were used in this study following approval by the institution’s Animal Care and Use Committee. The animals were maintained individually in temperaturecontrolled (23”C), light-regulated (I 4L: 1 OD) rooms, provided with monkey chow (Agway, Elizabeth City, NJ) twice per day and with water available ad libitum. The monkeys were evaluated daily for evidence of menses, with the onset of menses defined as cycle Day 1 of the menstrual cycle. Blood samples were collected daily

643

Theriogenology

from animals under ketamine sedation (10 mg/kg, im) Serum 17 beta-estradiol (E,) determinations were made RIA kits (ICN Biomedicals, Los Angeles, CA). The FSH by heterologous RIA procedures developed for assay of using materials distributed by the National Hormone Program, NIH (Bethesda, MD). Healthy male cynomolgus individually caged and maintained Ovarian

Reserve

In the (Lupron; TAP im) at 0630 h was drawn at 6. During the hyperstimlation Superovulation

monkeys were used as described above.

and serum stored at -20°C. using commercially available concentration was measured macaque gonadotropins (6), and Pituitary Distribution

as sperm

donors

and

were

Testing

initial menstrual cycle of the study, the GnRHa, leuprolide acetate Pharmaceuticals, Abbott Park, IL), was administered on Day 3 (100 IU, immediately following that day’s blood collection. Subsequently, blood 1600 h on cd 3, 0630 h and 1600 h on cd 4, and 0630 h on cd 5 and following menstrual cycle, all the animals underwent controlled ovarian as described below. and Oocyte

Collection

Beginning on Day 2 of menses, female monkeys were given 25 IV im human FSH (hFSH;hFSH/25 IU human LH (Pergonal, Serono) from Days 7 to 9. Half of the total daily hormone dose was given after blood sampling (0600 to 0800 h), and the remainder was given between 1500 and 1600 h. Ovarian hyperstimlation was discontinued in animals not demonstrating overall increases in estrogen production over the first 6 d of treatment. On Day 9, or when E, values reached 1000 pg/ml, the monkeys were given a single injection of hCG (1000 IU Profasi, Serono, Randolph, MA) to induce final oocyte maturation. Follicles were aspirated 32 and 34 h after administration of hCG. Follicular contents were aspirated into sterile tubes containing 1 .O ml of TALP-HEPES (2) culture medium containing 0.3% bovine serum albumin (BSA) and heparin (5 IU/ml). Following identification, oocytes were transferred to equilibrated TALP (2) medium containing 0.3% BSA. Evaluation of nucleus maturity was based on that previously described for the monkey (9). In Vitro

Fertilization

Monkey semen was collected by rectal electroejaculation under ketamine sedation (IO mg/kg, im). Upon collection, the semen sample was mixed with 3.0 ml TALP, washed twice by centrifugation (7 min, 105 x g), and filtered through a glasswool column (14) to separate motile from nonmotile spermatozoa. Analysis of sperm number and motility was performed on the filtered sample, which was resuspended to a concentration of 5 x IO6 cells/ml and stored at 37”C, 5% CO,. Prior to use for insemination, the spermatozoa were exposed to 1 mM dibutyl cyclic adenosine 3’-5’monophosphate and 1 mM caffeine (2) and incubated at 37”C, 5% CO2 for 1 h.

644

Theriogenology

Oocytes were cultured (37”C, 5% CO,) in TALP medium until extrusion of the first polar body, at which time they were inseminated with 5 x IO4 motile sperm/ml. After 6 to 8 h of culture with spermatozoa, the oocytes were examined at x 200 magnification with Hoffman optics for the presence of pronuclei. Statistical

Analysis

Th means and standard deviations of FSH at Day 3 of the menstrual cycle were analyzed by unpaired t-tests. The change in estrogen from Day 3 to Day 4 following GnRHa administration was analyzed by repeated measures analysis of variance (ANOVA). In all cases, an .alpha level of P < 0.05 was considered significant. Discriminate analysis was used to classify aspiration of subjects as successful or unsuccessful based on the values of the marker variables. RESULTS Of the 26 monkeys studied, a poor response to control ovarian hyperstimulation was encountered in 9 (34.6%) animals and the stimulation protocol was terminated. In these animals, the mean E, on cycle Day 6 of stimulation was 66.2 pg/ml (range 37 to 121) compared with 267.2 pg/ml (range 113 to 643) on Day 6 in the 17 animals taken to the stage of follicle aspiration. Of the animals taken to aspiration, a total of 131 preovulatory oocytes were collected, with a mean of 7.7 oocytes (range 2 to 26) per animal. One hundred and twenty-one oocytes were inseminated and 80 (66.1 %) fertilized. The variables analyzed for markers of successful stimulation were Day 3 of the cycle FSH concentration and the change in E, from Days 3 and 4 following GnRHa administration. The distributions of Day 3 FSH levels for successful (mean + SD 17.9 + 5.3) and unsuccessful (17.2 + 5.2) stimulations were not significantly different (P = 0.769). Analysis of the estrogen response following GnRHa administration indicated that the increase in estradiol at 24 h was significantly greater for successful aspirations than for unsuccessful ones (P = 0.0419). The mean change in estrogen for the successful monkeys was 97.8 pg/ml(95% CL = 49.7-l 45.9 pglml), whereas the mean change was only 21.6 pg/ml (95% CL = -5.5-48.6 pg/ml) for the Therefore, the estrogen increment following GnRHa unsuccessful animals. administration has the potential to distinguishsuccessful and unsuccessfulaspirations. Given the differential response to GnRHa stimulation, discriminant analysis was used to determine a model to discern between successful and unsuccessful stimulations. The analysis results in a “discriminant function” that allows for the classification of a subject as a success or failure based on knowledge of the marker variable, in this case, the change in E, following the GnRHa stimulation test. A regression-like equation was then calculated for each group. For the successful aspiration group, the equation is

Theriogenology

645

y = -1.33903 while

that

+ 0.01321

of the unsuccessful

y = -0.72455

+ 0.00291

x E, change aspiration

group

is

x E, change.

Both equations are solved for a given subject’s value of E, change, and the animal is then classified into the group with the largest value of y. As demonstrated in Table 1, the differential response of E, production after GnRHa treatment was used to correctly identify 78% of the animals subsequently canceled for poor ovarian response, but only 65% of those proceeding to oocyte retrieval. Future assignment to the aspiration versus treatment canceled group can be made according to which equation produces the larger value. Table

Actual

1.

Ability of the GnRHa stimulation test to identify good and poor responses to control ovarian hy‘perstimulation in the monkey model

Predicted

Asoiration

Asoiration

Yes

No

Yes

11

6

65

No

2

7

78

13

13

69

Total

% Correct

DISCUSSION In both the human (8) and monkey(g), the success of in vitro fertilization is strongly influenced by the number of mature oocytes that can be obtained following controlled ovarian hyperstimulation. It has been shown in the human that a female’s age has a strong influence on this ovarian response, resulting in an age-associated increase in IVF cancellation rates (7,161. However, other markers, such as basal FSH, have been shown to be more sensitive than age in predicting IVF outcome (11). In humans, the first marker of diminished ovarian reserve is elevated basal (Day 3) FSH (I I,1 5). More recently, this marker has been shown to be superior to age in a large clinical experience (18). However, Day 3 FSH is an indirect method of evaluating ovarian reserve since it measures the amount of inhibin/E, produced by a cohort of follicles, the feedback sensitivity of the hypothalamus-pituitary axis, and the production of FSH by the pituitary (17). For this reason, the Day 3 FSH test may identify only extreme cases of poor response to control ovarian hyperstimulation.

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Theriogenology

To assess ovarian responsiveness more directly by measuring the size of the recruitable cohort of follicles, stimulation tests have been proposed which depend on the production of gonadotropins by the pituitary and the response of the ovary to stimulation. Among these, the GnRHa stimulation test (I 91 and the clomiphene citrate challenge test (12) have been described, and both have successfully uncovered additional cases in which limited ovarian response was subsequently observed. It appears that, among women, stimulation tests are more sensitive than basal testing alone. The need to predict ovarian responsiveness is heightened in the monkey model where the female’s age is not known with certainty and where only one cycle is possible due to the development of antibodies to human gonadotropin. For this reason, we examined a group of monkeys by both the static basal (Day 3) FSH and the GnRHa stimulation test. Evaluation of the data showed that basal FSH was not predictive of which monkeys were subsequently removed from treatment for inadequate ovarian response. We acknowledge that FSH assay utilized in the present study is a heterologous assay which may lack the discriminative capacity of the ultrasensitive human assays. However, to date this is the only practical way of quantitating FSH levels in non-human primates. The GnRHa stimulation test was able to detect 7 of the 9 cycles which were ultimately canceled, providing a positive predictive value of 78%. The GnRHa test was less successful at identifying cycles that ultimately went to oocyte retrieval, misclassifying 6 of 17, for a negative predictive value of 65%. If there had been no test, 9 or 26 monkeys could be said to have failed to proceed to aspiration. Of those animals predicted to respond well (n = 131, eleven went on the retrieval (85%). Of those monkeys predicted to respond poorly (n = 131, 6 responded well and 7 responded poorly. Thus, when the predicted outcome is a good stimulation, we have a higher chance of achieving the predicted outcome. However, when the predicted outcome is a poor stimulation, there is still an almost 50% chance of a good outcome. Currently, when the GNRHa stimulation assay shows only a small or no increase in E,, we do not routinely perform control ovarian hyperstimulation on such animals, Unfortunately, we have less since most of such females would be canceled. confidence in a high E, rise after GnRHa in predicting the ultimate ovarian response; in many such cases cancellation of treatment seems probable but cannot yet be predicted. REFERENCES 1.

2.

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