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BOR - Papers in Press, published online ahead of print July 7, 2004.
Biol Reprod 2004, 10.1095/biolreprod.104.030585
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BIOLOGY OF REPRODUCTION 71, 1591–1597 (2004)
DOI: 10.1095/biolreprod.104.030585
© 2004 by the Society for the Study of Reproduction, Inc.

Pregnancy

Implantation and Pregnancy Following In Vitro Fertilization and the Effect of Recombinant Human Relaxin Administration in Macaca fascicularis1

E.S. Hayes3,5, E.C. Curnow3,5, A.O. Trounson4,5, L.A. Danielson6, and E.N. Unemori2,7

Monash Institute of Reproduction and Development,5 Clayton, Victoria 3168, Australia Department of Pathology,6 University of New Mexico School of Medicine, Albuquerque, New Mexico 87131 BAS Medical, Inc.,7 San Mateo, California 94402


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Implantation and early pregnancy, and the potential effects of the reproductive-hormone relaxin, were examined in the cynomolgus macaque (Macaca fascicularis) following in vitro fertilization and embryo transfer. Mature oocytes were collected from regularly cycling, female cynomolgus monkeys subjected to ovarian superovulation using recombinant human FSH and hCG. Oocytes fertilized in vitro were cultured to the 4- to 8-cell stage, slow-cooled, and stored in liquid nitrogen before thawing and embryo transfer. Regularly cycling recipients were administered recombinant human relaxin or vehicle for 21 days through the peri-implantation period (Day 0 = pump implantation), during which time the thawed embryos were transferred (Day 7). Endometrial thickness and the number of gestational sacs were monitored by ultrasound at three time points (Days 7, 21, and 28). The number of days of placental sign (implantation bleeding) in pregnant females and menses in nonpregnant females were also recorded. Implantation (gestational sacs/embryo transferred) and multiple pregnancy (multiple gestations/ pregnant recipient) rates were slightly higher in relaxin-treated recipients compared to vehicle-treated recipients. Administration of relaxin was associated with increased implantation bleeding in pregnant females. Endometrial thickness was increased in relaxin-treated recipients at Days 7 and 28 compared to Day 0, but these differences were not observed at the same time points in vehicle-treated females. Systemic administration of recombinant human relaxin in an in vitro fertilization/embryo transfer setting was associated with effects consistent with a role for this hormone in endometrial physiology in primates.

assisted reproductive technology, in vitro fertilization, pregnancy, relaxin, uterus


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
During their childbearing years, the reproductive-hormone relaxin is secreted into the circulation by the corpus luteum during the menstrual cycle and at elevated levels upon luteal rescue and throughout pregnancy [1]. Relaxin is also produced locally in the endometrium [2, 3], as is the receptor for this hormone [4–6], suggesting a potential autocrine or paracrine, as well as endocrine, influence of relaxin. In vitro, relaxin induces the expression of several molecules associated with implantation, including vascular endothelial growth factor (VEGF) [7, 8], insulin-like growth factor binding protein (IGFBP)-1 [9], glycodelin [10], and prolactin [9] in human endometrial cells. Glycodelin and IGFBP-1 are among the most upregulated genes when endometrial phenotype during the window of implantation is compared with phenotype during the late proliferative [11] or early secretory [12] phases by microarray analysis. The fact that serum [1] and endometrial [2, 3] relaxin are present during this period, that relaxin receptors are present in the endometrium during this time [4–6], and that relaxin is markedly upregulated on conception [1] strongly suggest a role for this hormone in regulating endometrial phenotype during the peri-implantation period and early pregnancy.

Elucidation of the potential role for relaxin during pregnancy in humans has been hampered by the dissimilarity in relaxin expression between primates and the more readily studied rodent species [13]. In rats and mice, circulating relaxin is absent during the estrous cycle and early pregnancy [14]. Instead, relaxin is first detectable in the serum at midpregnancy, with levels reaching a maximum in a prepartum surge immediately before or during delivery, which is consistent with a role for this hormone in tissue remodeling in preparation for birth in these species. Consistent with this expression pattern, the absence of relaxin in mice genetically deficient in relaxin (Rlx –/–) appears to have no discernible effect on early pregnancy in this species [15]. In contrast to rodents, women experience a rise in relaxin levels immediately after conception, with maximum concentrations being measurable in the first trimester of pregnancy; no prepartum relaxin surge occurs in women [16, 17].

Studies in some nonhuman primates have demonstrated enough fidelity with human physiology to suggest that they are reasonable to study as models of early pregnancy. Endocrine signals and the timing of implantation in the Old World cynomolgus monkey (Macaca fascicularis) are quite similar to those seen in humans [18]. Ovarian hormone production during the luteal phase of the cycle is similar in the macaque and humans, and both cycles end with sloughing of the endometrium. Relaxin levels in the circulation during the luteal phase and peri-implantation period approximate 50 and 40 pg/ml in humans and macaques, respectively [1, 18]. Implantation occurs 9 days after fertilization in the macaque [19], versus approximately 2 days earlier in humans [20], but trophoblast rescue of the corpus luteum occurs similarly in both species, with increased chorionic gonadotropin production 3 days after implantation. Enhanced relaxin and progesterone release by the corpus luteum, as well as increased estrogen secretion by the ovary, occur in response to conception in both M. fascicularis and humans. Relaxin levels approximating 1 ng/ml have been reported in pregnant females in both species [1, 18, 21]. Finally, the relaxin receptor, LGR7, has been detected in the endometrium of both the human and the cynomolgus monkey [5, 6].

The vagaries of mating and natural conception in macaques that would make the study of implantation and early pregnancy difficult can be circumvented by the use of in vitro fertilization (IVF) and embryo transfer (ET) techniques. Previous studies have shown some success in M. fascicularis, with implantation rates of 8.3%–12.0% having been achieved using one to four fresh or frozen-thawed embryos transferred per recipient into natural cycles [22– 24]. The present study was designed to investigate implantation and early pregnancy success in this species with IVF/ ET using more recently developed techniques [25–28]. The potential role of relaxin in early pregnancy was investigated under the controlled conditions of IVF/ET and, to our knowledge, represents the first report of the effects of administration of purified recombinant human relaxin (rhRLX) during implantation and early pregnancy in Old World monkeys.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Except where otherwise indicated, chemicals were obtained from Sigma Chemical Co. (St. Louis, MO). All experimental procedures were conducted at Monash University embryology facilities located at Bogor Agricultural University, Bogor, Indonesia. Experiments were approved by the Institutional Animal Care and Use Committee at Bogor Agricultural University (National Institutes of Health approval no. A5287-01). Male and female animals were anesthetized with ketamine (25 mg/kg i.m.) in combination with pre- and postoperative analgesia: ketoprofen (7.5 mg/kg i.m.) and butorphanol (0.15 mg/kg i.m.).

Oocyte Collection and Preparation

Sexually mature M. fascicularis females were monitored for regularity of three cycles before oocyte collection. Oocyte donors were subjected to ovarian superovulation with recombinant human FSH (60 IU/day i.m.; Serono Pty Ltd., Sydney, NSW, Australia) starting 2 days after the beginning of menses and continuing for 11–13 days [26]. Follicular maturation was completed with administration of hCG (urinary hCG; 1000 IU i.m.; Serono) the day following the last FSH administration. Ovarian development was monitored by ultrasound (5.0-MHz transabdominal curved array probe; Sonosite, Bothell, WA). Oocytes were retrieved from anesthetized animals at laparotomy 28–30 h following hCG administration. Follicles were aspirated at a negative pressure of 120–125 mm Hg using sterile ovum aspiration kits (Cook IVF, Brisbane, QLD, Australia).

Harvested oocytes were treated with hyaluronidase (80 IU/ml) to remove expanded cumulus cell populations and then classified according to nuclear maturation status (germinal vesicle, metaphase I, or metaphase II [MII]). The MII oocytes were placed into microdrops (50 µl) of human tubal fluid culture medium (HTF; Chemtec Pty Ltd., Melbourne, VIC, Australia), supplemented with 3.0 mg/ml of BSA (HTF+BSA), and overlaid with mineral oil. Oocytes were allowed to rest in culture medium for 4–6 h after collection before IVF [26].

Sperm Collection and Preparation

Epididymal sperm was collected from 16 anesthetized males by needle biopsy and transferred into Hepes-buffered HTF (mHTF; Chemtec), supplemented with 3.0 mg/ml of BSA (mHTF+BSA). Sperm were washed in mHTF+BSA and centrifuged for 10 min at 700 x g. The supernatant was discarded, and the sperm pellet was overlaid with fresh mHTF+BSA. A motile sperm-rich fraction was obtained by allowing sperm to swim-up into fresh mHTF+BSA for 15 min. Motile sperm were transferred to HTF+BSA in an atmosphere of 5% CO2 in air at 37°C and then subjected to activation with dibutyryl-cAMP (0.1 mM) and caffeine (0.5–1.0 mM) for 60 min before IVF.

IVF and Embryo Freezing

Oocytes were fertilized in vitro by adding aliquots of hyperactivated sperm (final concentration, 250 000 motile sperm/ml) to MII oocytes. Oocytes were incubated in the presence of sperm for 12–15 h in an atmosphere of 5% CO2 in air before transfer into fresh HTF+BSA. Fertilization was recorded as the presence of two pronuclei and second polar body extrusion. Zygotes were cultured in vitro in HTF+BSA at 37°C in an atmosphere of 5% CO2, 5% O2, and 90% N2. Embryos reaching the 4- and 8-cell stages of development were frozen, according to methods described previously [27], so that ET could be timed to the expected window of ovulation in recipient females. Briefly, embryos were initially placed into PBS, supplemented with 4 mg/ml of BSA (PBS+BSA), for 10 min at room temperature. Embryos were then transferred to a solution containing 1.5 M 1,2-propanediol (PrOH) in PBS+BSA for a further 10 min. Embryos were then transferred into a solution containing 1.5 M PrOH and 0.1 M sucrose in PBS+BSA and immediately loaded into prerinsed, 0.25-ml freezing straws (two embryos per straw). Straws containing embryos were heat-sealed and transferred to a controlled-rate freezing machine with a starting temperature of 20°C. Embryos were cooled at –2°C per minute to –6°C and then seeded. After being held at –6°C for 10 min, embryos were cooled at –0.3°C per minute to –40°C, then plunged directly into liquid nitrogen and stored.

Embryo Recipients and ET

Recipient females were monitored for regularity of three cycles before ET and were randomized to two groups before osmotic pump implantation. Pumps (model 2004; Alza Corporation, Palo Alto, CA) were used for delivery of rhRLX (lot 63601; Connetics Corporation, Palo Alto, CA) at a dose of 8 µg kg–1 day– or vehicle alone (25 mM acetate, pH 5.5) for 21 days. The rhRLX dose was selected on the basis of previously conducted clinical studies in humans [29] as well as a small pharmacokinetic study in cynomolgus monkeys, both of which confirmed that this dose would result in steady-state circulating relaxin levels of 1–2 ng/ml (data not shown). These levels approximate concentrations observed in pregnant women and macaques, which range from 0.03 ng/ml during the early luteal phase to 1.0 ng/ml during early pregnancy [1, 18, 21]. Pumps were loaded and primed at 37°C, according to the manufacturer's instructions. Proper loading of osmotic pumps was verified by weighing, according to the manufacturer's instructions (mean fill efficiency, 98.9% ± 0.8%, mean ± SD).

Pumps were implanted (Day 0) s.c. on the backs of recipient monkeys, under anesthesia, 7 days before ET (Day 7). Eleven females were dosed with rhRLX, and 11 received vehicle alone. Embryos produced through IVF were thawed according to established methods [23] and placed in culture medium (HTF+BSA) before ET. Four viable embryos (>50% blastomere survival postthaw) were transferred to each of the 22 recipient females during the expected ovulation window in a natural cycle (Days 13–17), which has been described previously [30]. Briefly, the ratio of the day of ovulation to the cycle length was used to identify the expected midpoint of the ovulation window, to which 2 days were added to determine the date of ET. Embryos were transferred at laparotomy through the fimbria into the midampullary region of each fallopian tube (n = 2 per oviduct) using sterile ET catheters (Cook IVF). Some of the thawed embryos were analyzed for development in vitro following culture in HTF+BSA.

Endometrial Thickness and Embryo Implantation

The aim of the present study was to assess the effects of systemic exposure to exogenous, highly purified, rhRLX during the peri-implantation period on implantation and pregnancy rates and other measures of endometrial function. Endometrial variables were assessed and analyzed by an individual (E.H.) who was blinded to the treatment groups. Endometrial thickness was monitored by ultrasound (5.0-MHz transabdominal curved array probe; Sonosite) in the transverse and sagittal planes on Days 0, 7, 21, and 28 following pump implantation. Assessments were considered to be reliable when two measurements could be obtained in each plane. Measurements in both planes were used for statistical analysis, and mean values are reported in Results. The number of gestational sacs and fetuses were recorded at Days 21, 28, and 67. Placental size (circumference and surface area) was also monitored by ultrasound at Day 67. Pregnancies were terminated following the Day 67 determination of placental size.

Implantation rate per treatment group was reported as the number of embryos implanted/total number of embryos transferred; in the present study, the total number of embryos transferred per treatment group was 44. Pregnancy rate was expressed as the percentage of pregnant recipients/ total number of recipients. Multiple pregnancy rate was expressed as the percentage of multiple gestations/number of pregnant recipients.

Implantation bleeding, or placental sign, which is a phenomenon associated with embryonic implantation in nonhuman primates [31], was quantified by monitoring the first day of observance of bleeding sign (start) and the total number of days of visible vaginal bleeding following ET (total) in relaxin- and vehicle-treated pregnancies. Menses, which occurred when ET did not result in a pregnancy, was also calculated as the number of days of visible vaginal bleeding.

Serum Hormone Analysis

Serum rhRLX levels were measured by quantitative ELISA specific for human relaxin (H2), as reported in detail elsewhere [32]. Samples were initially diluted 1:2 and compared against a standard curve (11.72–750 pg/ ml, n = 7, r = 0.99, t = 161.5, P < 0.0001). Samples that produced readings above the standard curve were diluted 1:10 and run a second time. Concentrations were corrected for dilution factor. Intra- and interassay coefficients of variation were 4.2% and 7.2%, respectively. Serum hormone values were log transformed before statistical analysis. Analyses performed before this study demonstrated that cynomolgus monkey relaxin at levels observed during normal pregnancy (~1 ng/ml) were less than the limit of detection in this assay (data not shown).

Data Analysis

Data for endometrial thickness, implantation bleeding, and placental sizes (circumference and surface area) were expressed as mean ± SEM (n = 4–11 animals). Significance of treatment effects on endometrial thickness or serum rhRLX levels was assessed by a Student t-test following two-way ANOVA with one repeated measure [33]. If the two-way ANOVA indicated an effect of time (number of days) on thickness or serum rhRLX levels, the paired t-test was used to make pairwise comparisons following a one-way ANOVA. Significant differences in implantation bleeding and placental size were determined using a Student t-test [33]. Significant differences in implantation and pregnancy rates were determined by chi-square test, except for analysis of multiple pregnancy rates on Day 67, for which the Fisher exact test was used [33]. Differences were considered to be significant when P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Donor Characteristics and Embryo Production

Donor female cycle interval (mense to mense) and duration of menses in the three cycles preceding the donor cycle were regular, with cycle intervals ranging from 31.3 ± 0.7 days to 33.9 ± 2.9 days and menses durations ranging from 2.9 ± 0.2 days to 3.7 ± 0.3 days. A total of 413 oocytes from 10 females were aspirated, and 340 oocytes were classified as MII and suitable for use in IVF procedures. Of the MII oocytes inseminated, 222 fertilized (60% ± 12%) and 212 fertilized embryos cleaved to the 8-cell stage within 48 h of insemination (95% ± 3%). Fertilization and cleavage rates, adjusted for failed fertilizations resulting from poor sperm quality, poor response of sperm to chemical hyperactivation, and/or poor oocyte quality (2/10 superovulation cycles), were 80% ± 8% and 95 ± 4%, respectively.

Recipient Treatment and ET

Recipient cycle interval and duration in the three cycles preceding pump implantation were regular, with cycle intervals ranging from 29.4 ± 0.8 days to 32.6 ± 1.6 days and cycle durations ranging from 3.6 ± 0.2 days to 3.6 ± 0.3 days. Body weights of the recipients were similar at pump implantation (vehicle-treated group, 2.65 ± 0.31 kg; rhRLX-treated group, 2.81 ± 0.36 kg) and at ET 7 days later (vehicle-treated group, 2.67 ± 0.35 kg; rhRLX-treated group, 2.77 ± 0.32 kg).

Embryos transferred to rhRLX- and vehicle-treated animals exhibited postthawing cell survival rates of 90% ± 3.3% and 89% ± 3.1%, respectively. The first and second embryos transferred to the left and right oviducts of rhRLX- and vehicle-treated animals did not differ significantly in their cell survival rates, with postthawing cell survival rates ranging from 86.3% ± 6.2% to 97.7% ± 2.3%. All the thawed embryos exhibiting 50% cell survival postthawing (not suitable for transfer) developed to hatched blastocysts (4/4, 100%) in culture. Embryos with less than 50% blastomere survival postthawing failed to develop past the 8-cell stage (0/3, 0%).

Serum Hormone Analysis

None of the animals in either the vehicle- or rhRLX-treated groups had detectable rhRLX levels on Day 0 before pump implantation. The rhRLX was not detected in any of the vehicle-treated animals at any of the observation time points (Day 7, 21, 28, or 67). In the rhRLX-treated group, 10 of 11 animals demonstrated circulating rhRLX levels at Day 7. The average Day 7 serum concentration in these animals was 1.2 ± 0.37 ng/ml (n = 10). The single monkey that did not have detectable rhRLX levels on Day 7 failed to demonstrate rhRLX levels at any of the observation time points; therefore, this female was omitted from the rhRLX-treated group and all the analyses presented below. At Day 21, rhRLX levels in the rhRLX group averaged 3.7 ± 1.3 ng/ml (not significant compared to Day 7). By Day 28 and Day 67, serum rhRLX concentrations had dropped to 0.20 ± 0.10 ng/ml and 0.04 ± 0.04 ng/ml, respectively (P = 0.035 and 0.03 vs. Day 21, respectively).

Endometrial Thickness and Embryo Implantation

Four endometrial thickness measurements were technically possible on all four sonograph days (Days 0, 7, 21, and 28) in seven females in the vehicle-treated group and in nine females in the rhRLX-treated group. A significant effect of treatment and time on endometrial thickness was found (P < 0.0001 for interaction). Comparisons between mean thickness measurements in the vehicle (n = 7) and treatment (n = 9) groups at the observation time points indicated that mean thicknesses in the vehicle and rhRLX groups on Day 7 were significantly different (P = 0.01) (Table 1). No significant time effect for vehicle controls was found (P = 0.10), but a significant time effect for the relaxin group was evident (P < 0.0001). In this group, endometrial thickness on Day 7 measured 0.32 ± 0.02 cm, a significant increase in thickness from Day 0 (0.23 ± 0.01 cm, P = 0.003). Whereas all nine females in the rhRLX-treated group showed an increase in endometrial thickness from Day 0 to Day 7, five of the seven in the vehicle-treated group showed increases, one showed no change, and one showed a decrease in thickness (Fig. 1). The rhRLX treatment-associated increase in thickness was transient: By Day 21, the difference was not significant, and on Day 28, the difference was smaller, when compared to Day 0 (P = 0.035).


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  		TABLE 1. Serial endometrial thickness measurements in vehicle- and rhRLX-treated recipient Macaca fascicularis.



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  		FIG. 1. Change in endometrial thickness between Day 0 and Day 7 in vehicle- and rhRLX-treated females. Two endometrial thickness measurements were made in each of the sagittal and transverse planes for each female and then averaged to yield one thickness measurement for each female on each of Days 0 and 7. Thickness increased in all females in the rhRLX group (P = 0.003, paired t-test), whereas it increased in five of the seven females in the vehicle-treated group (not significant)

Of the 44 embryos transferred to 11 vehicle-treated recipients, 13 implanted by Day 21, for an implantation rate of 29.5% in the vehicle group (Table 2). This was lower than the implantation rate of 42.5% (17/40 embryos) observed in the rhRLX-treated group at Day 21. Implantation rates in the rhRLX-treated group were also higher than those observed in the vehicle-treated group at Days 28 and 67. However, these differences did not reach statistical significance. Overall pregnancy rates at Day 21 were high in both groups: 91% (10/11) in the vehicle-treated group, and 80% (8/10) in the rhRLX-treated group. Sustained pregnancy rates at Day 67 were 54.5% (6/11) in the vehicle-treated group and 50.0% (5/10) in the rhRLX-treated group. Although three multiple pregnancies (three twin pregnancies) initially occurred in the vehicle-treated group (3/9, 33%), none remained at Day 67 (0/6, 0%). The number of multiple pregnancies in rhRLX-treated animals (three twins, three triplets) was initially six (6/8, 75%), of which three remained at Day 67 (3/5, 60%; P = 0.06 vs. vehicle-treated pregnancies at Day 67). Of these three remaining pregnancies, two were twin pregnancies, and one was a triplet pregnancy.


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  		TABLE 2. Implantation and pregnancy rates in vehicle- and rhRLX-treated Macaca fascicularis.

In pregnant females of the vehicle-treated group (n = 6), placental sign-associated bleeding started 19.7 ± 2.3 days following ET and lasted an average of 9.8 ± 2.6 days. In rhRLX-treated pregnancies (n = 5), implantation bleeding started 12 ± 0.8 days after ET (P < 0.02 vs. vehicle group) and lasted an average of 19.2 ± 2.2 days (P < 0.05 vs. vehicle-treated pregnancies). Menses in nonpregnant females (n = 4) in the vehicle-treated group occurred at Day 20.0 ± 3.8 following ET and lasted 3.3 ± 0.3 days, with one additional female showing no evidence of menses. Menses initiated 16.2 ± 1.5 days following ET in nonpregnant rhRLX-treated females and lasted for 4.0 ± 0.3 days (n = 5; not significant compared to vehicle-treated group).

At Day 67, assessment of placental surface area and circumferences by ultrasound was technically feasible in four of the six control pregnancies and in three of the five rhRLX-treated pregnancies. Fetal movement interfered with measurement in the other females. Placental surface area was 0.59 ± 0.17 cm2 and 0.87 ± 0.18 cm2 in the vehicle- and rhRLX-treated groups, respectively. Placental circumference measured 2.73 ± 0.68 cm and 3.37 ± 0.33 cm in the vehicle- and rhRLX-treated groups, respectively. Neither of these differences was statistically significant.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present study demonstrates excellent IVF success rates in M. fascicularis utilizing current techniques, with fertilization and cleavage rates of 80.4% and 95.0%, respectively. Because of the cost and restricted availability of macaques, one of the goals of the present study was to examine implantation rates utilizing as few recipients as possible. In this effort, four embryos were transferred to each recipient, and implantation and pregnancy rates were determined over 67 days. An implantation rate of 29.5% was achieved in the control (vehicle-treated) animals, which is much higher than that observed in earlier studies [22–24]. A pregnancy rate of 91% was achieved at Day 21 in the macaques, and at Day 67 (of what is typically a 165-day gestation), an ongoing pregnancy rate of 54.5% was observed. These results demonstrate the feasibility of using M. fascicularis for the study of implantation and early pregnancy as well as of IVF/ET. The similarities between M. fascicularis and humans in relaxin and LGR7 expression during the reproductive cycle and pregnancy [1, 4–6, 18– 21] also form the foundation for future investigations using this model to study the importance of relaxin during early pregnancy in humans.

The present study also aimed to detect the effects of systemic administration of exogenous, highly purified rhRLX, specifically during the peri-implantation period, on implantation and pregnancy rates and other indirect measures of endometrial function during this period. Because the processes of implantation and early pregnancy are extremely sensitive to external perturbations, no endometrial biopsies were performed; therefore, what potential cellular or tissue changes were induced by rhRLX in M. fascicularis is a question to be answered in future studies. rhRLX was infused from 7 days before ET to 14 days post-ET in the present study, encompassing the luteal phase and extending into early pregnancy. The biological activity of human rhRLX on nonhuman primate endometrium has been demonstrated previously in vitro [34]. A previous study of hormone levels during early pregnancies following ET in natural cycles and naturally mated cycles in macaques found an association of the rise in relaxin and CG levels with maternal recognition of the trophoblast [35]. An average delay of 3 days in the elevation of relaxin and CG was found in ET cycles relative to spontaneous pregnancies, suggesting that the failure of some pregnancies following ET could result from the inability of the maternal system to provide adequate support for the implanting embryo. Although the present study did not specifically address this issue, the provision of exogenous rhRLX during this period may have contributed to the slightly enhanced implantation rate.

The fact that recipient monkeys were presumably fertile at the outset and may have exhibited normal levels of endogenous macaque relaxin were admittedly factors that could have biased the present study toward not showing a more pronounced effect of exogenously provided rhRLX on implantation and pregnancy rates. However, finding a cohort of infertile or relaxin-deficient monkeys in which to perform the present study was unrealistic, so it was performed with this caveat in mind. A relaxin-ablation experiment, in which both circulating and endometrial relaxin could be neutralized, would be ideal to demonstrate the importance of relaxin during this period.

Consistent with pharmacokinetics observed in humans [29], the infused dose of 8 µg kg–1 day–1 of rhRLX produced serum levels on Day 7 that were similar to concentrations observed in human and macaque pregnancy (~1 ng/ml) [1, 18, 21]. Although not statistically significant, a small rise in mean serum rhRLX levels was observed from Day 7 to Day 21 in rhRLX-treated animals, and this is believed to result from an antibody-mediated delay in clearance of rhRLX (unpublished observations). Antibodies directed against human rhRLX have been observed in response to relaxin administration in other monkey species [36] and is an unavoidable consequence of treatment with a heterologous molecule. The functional significance of the elevated rhRLX levels with respect to the observed experimental outcomes is not known.

The infusion of rhRLX was associated with a 44% increase in implantation rate in the rhRLX group compared to the control group, but this difference did not achieve statistical significance. Power calculations based on the observed differences in implantation rate indicate that a sample size of at least 120 embryos (30 monkeys) per group would be required to achieve statistical significance (P < 0.05). Maintenance of a multiple pregnancy (twins or triplets) in 60% of pregnant rhRLX-treated females over 67 days in a species that exhibits very low rates (0.1%) of multiple gestations in natural and captive-breeding environments [37] also suggests a possible influence of rhRLX. The use of ultrasonography to examine early pregnancies in macaques has led to the conclusion that the occurrence of twin gestations is greater than the frequency of live-born twins, which is similar to the "vanishing twin" phenomenon observed in humans [38]. In the present study, multiple gestations comprised 67% and 60% of pregnancies at Day 21 and Day 67, respectively, in the rhRLX-treated females and 30% and 0%, respectively, at the same time points in the vehicle-treated monkeys.

Positive effects from the infusion of rhRLX on endometrial thickness were observed in the present study. In humans, endometrial thickness increases progressively during a normal menstrual cycle, from a nadir in the early proliferative phase to a maximum during the early/mid-luteal phase and then remaining at this maximum until 1 or 2 days before menses [39]. This increase is believed to reflect physiological alterations occurring in the tissue, including an increase in endometrial blood flow [39, 40] and glandular secretory and stromal predecidual changes [41] that occur under the influence of the hormonal milieu. Thus, thickness measurements may be useful as a general descriptive feature of a normally developing endometrium and, in fact, have been used to identify abnormally thin endometria, which for whatever reason have a high implantation failure rate following IVF [42, 43]. Our results demonstrate that infusion of exogenous rhRLX had a statistically significant, positive influence on the thickness of the endometrium in cynomolgus monkeys. The increase in thickness may result from the concerted effects of rhRLX on multiple cell types within the endometrium, because both glandular and stromal elements possess relaxin receptors [4, 5, 6].

rhRLX administration in cynomolgus monkeys was also associated with an increased placental sign, or implantation bleeding. Placental sign is a natural phenomenon that appears several days following mating in pregnant females in some nonhuman primate species [31]. The bleeding is believed to be associated with embryonic implantation in monkeys and, contrary to the situation in humans, is a positive sign of pregnancy. rhRLX treatment was associated with a significantly shorter time period to first appearance of the placental sign and a significant increase in the duration of implantation bleeding in M. fascicularis compared to treatment with vehicle alone. A reasonable hypothesis for the increases in placental sign, as well as in endometrial thickness, is that rhRLX has a positive influence on endometrial perfusion, which increases in the luteal phase of the menstrual cycle [44] when relaxin is naturally present. Observations made previously in the course of conducting clinical trials concerning systemic sclerosis have suggested that relaxin has an effect on uterine blood flow, because administration of rhRLX was associated with an increase in menstrual bleeding in a large proportion of relaxin-treated women [7, 29, 45, 46]. Although only relatively impure porcine relaxin was available at the time [13], a key study performed in the 1950s associated administration of relaxin with a histologically apparent increase in blood vessels in the endometrium in monkeys [47, 48]. More recently, an increase in the number of arterioles was found in the endometrium in response to administration of human rhRLX in rhesus monkeys [49].

These observations made following relaxin administration in humans and nonhuman primates are consistent with effects of relaxin on gene expression in endometrial target cells. For example, rhRLX caused a dose-dependent increase in the secretion of the cytokine VEGF in normal human endometrial stromal and epithelial cells in vitro [7, 8]. Both VEGF and its receptors, which are expressed in the primate endometrium [50–52], may be at least partly responsible for increased blood vessel growth, vasodilation, and vascular permeability that occur in the luteal phase of the menstrual cycle [52–56]. Although the highest peaks of VEGF expression and endothelial proliferation occur in the proliferative phase of the cycle, the steady rate of vessel growth during the mid- to late-luteal phase, accompanied by continual stromal and epithelial production of VEGF, is believed to be important in implantation [52, 56]. The process of stromal cell decidualization induces both VEGF [57, 58] and relaxin [57], suggesting that both are important in implantation. Although VEGF is a potential downstream mediator of relaxin activity in the endometrium, others, including steroid receptors [59] or members of the endothelin system [60], also likely play a role. Indeed, the upregulation of the endothelin type B receptor has been implicated in relaxin-induced dilation of the renal [60] and coronary [61] vasculature. Interestingly, decidualization of endometrial stromal cells in vitro under conditions that induce relaxin and VEGF mRNA is also associated with marked upregulation of transcripts of the endothelin type B receptor [57].

In addition, the importance of VEGF [62] and proteinases [63] in the induction of menses suggest that relaxin may play a role in the menstrual cascade. Because angiogenesis, vasodilation, and the upregulation of certain matrix metalloproteinases, which occur in preparation for embryonic implantation, are the same factors that regulate menstrual bleeding in the absence of a conceptus, relaxin may also play a role in the regulation of menstruation. This is consistent with the finding of menorrhagia in a large proportion of women who were administered relaxin in clinical trials [7, 29, 45, 46].

In summary, the present study demonstrates that excellent IVF/ET success rates can be achieved in M. fascicularis and that it, like the rhesus monkey, is a suitable model for the study of implantation and early pregnancy in humans. Administration of human rhRLX to cycling females during the peri-implantation period resulted in a transient increase in endometrial thickness and increased implantation-related bleeding. These results are consistent with a role for relaxin in modulating endometrial phenotype during the implantation period in the primate. It remains to be determined whether pharmacological manipulation of the endometrium with rhRLX could be of value in human IVF settings where endometrial thickness and reduced endometrial vascularization have a negative impact on pregnancy rates [64]. Recently discovered relaxin receptors [65] offer the promise of further identification of specific relaxin targets within the uterus, as well as the design and development of potent and selective synthetic relaxin agonists and antagonists with which to perform functional studies. The use of these tools in nonhuman primate models, such as the one described here, should accelerate the elucidation of relaxin's role in early human pregnancy.


   FOOTNOTES
 
1 Funded by Connetics Corp., Palo Alto, California. Back

2 Correspondence: E.N. Unemori, BAS Medical, Inc., 1660 South Amphlett Blvd., Ste. 200, San Mateo, CA 94402. FAX: 650-235-3579; eunemori{at}basmedical.com Back

3 Current address: The Washington National Primate Research Center, University of Washington, Box 357331, Seattle, WA 98195 Back

4 Current address: Monash Immunology and Stem Cell laboratories, Monash University, Clayton, VIC 3800, Australia Back

Received: 5 April 2004.

First decision: 23 April 2004.

Accepted: 14 June 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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