Use of urinary 13,14, dihydro-15-keto-prostaglandin F2α (PGFM) concentrations to diagnose pregnancy and predict parturition in the giant panda (Ailuropoda melanolecua).

Pregnancy determination is difficult in the giant panda (Ailuropoda melanolecua), representing a challenge for ex situ conservation efforts. Research in other species experiencing pseudopregnancy indicates that urinary/fecal concentrations of 13,14, dihydro-15-keto-prostaglandin F2α (PGFM) can accurately determine pregnancy status. Our objective was to determine if urinary PGFM concentrations are associated with pregnancy status in the giant panda. Urinary PGFM concentrations were measured in female giant pandas (n = 4) throughout gestation (n = 6) and pseudopregnancy (n = 4) using a commercial enzyme immunoassay. Regardless of pregnancy status, PGFM excretion followed a predictable pattern: 1) baseline concentrations for 11-19 weeks following ovulation; 2) a modest, initial peak 14-36 days after the start of the secondary urinary progestagen rise; 3) a subsequent period of relatively low concentrations; and 4) a large, terminal peak at the end of the luteal phase. Pregnant profiles were distinguished by an earlier initial peak (P = 0.024), higher inter-peak concentrations (P < 0.001), and a larger terminal peak (P = 0.003) compared to pseudopregnancy profiles. Parturition occurred 23 to 25 days from the initial PGFM surge and within 24 hours of the start of the terminal increase. These pattern differences indicate that urinary PGFM monitoring can be used to predict pregnancy status and time parturition in the giant panda. Furthermore, this is the only species known to exhibit a significant PGFM increase during pseudopregnancy, suggesting a unique physiological mechanism for regulating the end of the luteal phase in the giant panda.

The acute phase protein ceruloplasmin as a non-invasive marker of pseudopregnancy, pregnancy, and pregnancy loss in the giant panda.

After ovulation, non-pregnant female giant pandas experience pseudopregnancy. During pseudopregnancy, non-pregnant females exhibit physiological and behavioral changes similar to pregnancy. Monitoring hormonal patterns that are usually different in pregnant mammals are not effective at determining pregnancy status in many animals that undergo pseudopregnancy, including the giant panda. Therefore, a physiological test to distinguish between pregnancy and pseudopregnancy in pandas has eluded scientists for decades. We examined other potential markers of pregnancy and found that activity of the acute phase protein ceruloplasmin increases in urine of giant pandas in response to pregnancy. Results indicate that in term pregnancies, levels of active urinary ceruloplasmin were elevated the first week of pregnancy and remain elevated until 20-24 days prior to parturition, while no increase was observed during the luteal phase in known pseudopregnancies. Active ceruloplasmin also increased during ultrasound-confirmed lost pregnancies; however, the pattern was different compared to term pregnancies, particularly during the late luteal phase. In four out of the five additional reproductive cycles included in the current study where females were bred but no birth occurred, active ceruloplasmin in urine increased during the luteal phase. Similar to the known lost pregnancies, the temporal pattern of change in urinary ceruloplasmin during the luteal phase deviated from the term pregnancies suggesting that these cycles may have also been lost pregnancies. Among giant pandas in captivity, it has been presumed that there is a high rate of pregnancy loss and our results are the first to provide evidence supporting this notion.

Pregnancy diagnosis in urine of Iberian lynx (Lynx pardinus).

Diagnosis of pregnancies is an important management tool for the Iberian lynx Conservation Breeding Program, a program geared to recover the world's most endangered felid. Non-invasive methods such as fecal hormone analyses are not applicable to the lynx, since fecal progestin does not follow the typical pregnancy pattern of felids. Therefore, we aimed to test whether urine can be used as an alternative substance for pregnancy diagnosis in the Iberian lynx. Progesterone immunoreactive metabolites were determined in urine samples of pregnant and non-pregnant females before and during breeding season. Additionally, we used the Witness Relaxin test to determine relaxin in blood and urine. No differences were found in progestin concentrations determined in urine samples collected from pregnant and non-pregnant animals between day 1 and 65 following mating. Although the Witness Relaxin test was positive in serum samples collected from animals between day 32 and 56 of pregnancy, it failed in both fresh and frozen urine samples collected from the same stage of pregnancy. A weak relaxin reaction in urine samples collected from animals between day 29 and 46 of pregnancy was detectable after urines were concentrated by ultrafiltration (>50x). Concentrated samples obtained from non-pregnant and early pregnant animals yielded negative test results. In conclusion, the Witness Relaxin test can be applied for pregnancy diagnosis in Iberian lynx in both serum and concentrated urine samples obtained during the second half of pregnancy. A positive relaxin test indicates an ongoing pregnancy, whereas negative tests must be judged carefully as hormone concentrations might be below detection thresholds.

Maternal factors modulate the increase in vasoactive substances during rat pregnancy.

OBJECTIVE: To explore if the changes in vasoactive substances observed during early pregnancy in the rat are modulated by maternal or fetoplacental factors. METHODS: Urinary excretion of cGMP, 6-keto-prostaglandin-F1 alpha (6-keto-PGF1 alpha), thromboxane B2 and kallikrein activity was measured in pregnant (P, n = 11), pseudopregnant (PSP, n = 12), and virgin (n = 13) rats and in ovariectomized virgin rats supplemented with slow-release pellets containing either progesterone (50 mg/pellet) or estradiol (0.5 mg/pellet) or a combination of both hormones, for 21 days. RESULTS: The cGMP excretion was higher in PSP rats than in virgin rats at day 5 (virgin = 82 +/- 7, P = 93 +/- 5, PSP = 110 +/- 8 nmol/24 h, p < 0.05); at day 10, values were significantly increased in P and PSP rats. 6-keto-PGF1 alpha excretion was similarly elevated in P and PSP rats at day 5 (virgin = 120 +/- 10, P = 160 +/- 10, and PSP = 174 +/- 14 ng/24 h, p < 0.01). This trend was still present at day 10. Thromboxane B2 excretion showed a nonsignificant increase in P and PSP rats in day 5; at day 10, values were significantly elevated in both experimental groups (virgin = 23 +/- 2, P = 32 +/- 4, and PSP = 32 +/- 2 ng/24 h, p < 0.05). Kallikrein excretion was significantly increased in PSP and P rats at days 5 and 10. Estradiol or progesterone administration caused a significant decrease in serum aldosterone and an increase in urinary kallikrein activity. CONCLUSIONS: These results indicate that during the first half of rat pregnancy, the increment in vasoactive substances is modulated by maternal and not by fetoplacental factors.

The human corpus luteum: reduction in macrophages during simulated maternal recognition of pregnancy.

It has been shown that immune cells, particularly macrophages, accumulate in the corpus luteum during luteolysis. This study aimed to investigate the effect of maternal recognition of pregnancy on the localization and numbers of macrophages in the human corpus luteum. Corpora lutea (n = 12) were obtained from normally cycling women at the time of hysterectomy and were dated on the basis of serial urinary luteinizing hormone (LH) estimation. In addition, corpora lutea (n = 4) were collected from women who had received daily doubling doses of human chorionic gonadotrophin (HCG) to mimic the hormonal changes of early pregnancy. Macrophages were localized by immunohistochemistry using an anti-CD68 antibody. Steroidogenic cells, steroidogenic cells of thecal origin and endothelial cells were identified on serial sections by immunohistochemistry for 3beta-hydroxysteroid dehydrogenase, 17alpha-hydroxylase and von Willebrand factor, respectively. The luteal cells capable of responding directly to HCG were identified by isotopic in-situ hybridization for messenger RNA encoding LH/HCG receptors. Macrophages were localized primarily to the vascular connective tissue and theca-lutein areas of the corpus luteum, although some were found in the granulosa-lutein cell layer. Macrophage numbers increased throughout the luteal phase to a maximum in the late-luteal phase (P < 0.05). Luteal 'rescue' with HCG was associated with a marked reduction in the numbers of tissue macrophages when compared with those of the late-luteal phase (P < 0.001). One of the effects of HCG during maternal recognition of pregnancy is to prevent the normal influx of macrophages into the corpus luteum. As LH/HCG receptors localized to the steroidogenic cells, this implies a fundamental role for steroidogenic cell products in the control of macrophage influx into the human corpus luteum.

Plasma level, urinary excretion, and metabolic production of cGMP during gestation in rats.

We postulated that guanosine 3',5'-cyclic monophosphate (cGMP), a cellular mediator of vascular smooth muscle relaxation, might mediate maternal renal and cardiovascular hemodynamic adaptation to pregnancy. Because extracellular levels of cGMP most likely reflect intracellular production, we began our investigation of this hypothesis by measuring the plasma concentration, urinary excretion, and metabolic clearance rates of cGMP during pregnancy in rats. Plasma cGMP was significantly elevated during mid- and late pregnancy, whereas urinary excretion of cGMP was increased throughout pregnancy. The fractional excretion of cGMP by the kidneys was 0.90 +/- 0.15 in the nonpregnant condition. In contrast, plasma levels of adenosine 3',5'-cyclic monophosphate were unchanged during pregnancy, and its urinary excretion rose slightly, reaching significance only on gestational day 20. There was also a significant rise in urinary excretion of cGMP throughout pseudopregnancy. The metabolic clearance rate of cGMP measured in chronically instrumented rats before, during, and after pregnancy was not significantly altered during gestation. The elevated plasma level of cGMP during gestation in rats, in the face of an unchanged metabolic clearance, reflects augmented tissue(s) production of cGMP, although enhanced cellular efflux may contribute. Because cGMP is a second messenger for several vasodilatory hormones, our data are consistent with the hypothesis that vascular production of cGMP may increase during pregnancy and thereby contribute to maternal renal and cardiovascular vasodilation. (Most investigators have not observed increment of plasma atrial natriuretic peptide in rat gestation; therefore this hormone is an unlikely first messenger for the elevated extracellular levels of cGMP that we have observed. Finally, pseudopregnant rats also showed enhanced urinary excretion of cGMP, which suggests that the proliferative activity that accompanies fetoplacental maturation, as well as hormones elaborated by the fetoplacental unit, is not necessary for the rise in urinary excretion of cGMP observed during pregnancy.(ABSTRACT TRUNCATED AT 250 WORDS)