Immunological detection of pregnancy: Evidence for systemic immune modulation during early pregnancy in ruminants.

Mammalian pregnancy creates unique challenges for immune systems highly evolved to detect and eliminate invading pathogens. Recognition of the challenges created by gestating a semi-allogeneic fetus evolved from the discipline of transplantation biology and were informed by studies on the unique natural parabiosis that occurs when female calves are gestated with twin male fetuses. These pregnancies typically result in an intersex female termed a freemartin, which revealed insights into development of the male and female reproductive tracts. However, they also uncovered important clues on immune tolerance with wide-ranging implications to reproductive biology, transplantation biology and autoimmune disease. Many studies focused on identifying mechanisms through which the fetus evades maternal immune detection and elimination. These included studies characterizing immune interactions between the fetus and mother at the nourishing interface of the placenta and uterine endometrium. This immunological forbearance only occurs under high concentrations of circulating progesterone. Beyond the requirement for progesterone, there has been considerable progress towards understanding the effects of conceptus signals on maternal immune function. One common theme is that pregnancy induces a T helper 2 immune bias as shown in several mammalian species, including domestic ruminants. However, a growing body of evidence shows that the fetus not only evades, but also provokes immune responses locally in the uterus and in peripheral tissues. This is perhaps most dramatically illustrated by domestic ruminants where the conceptus secretes a unique interferon in the opening salvo of hormonal communication with the maternal immune system. The role of interferon tau in regulating expression of genes of the innate immune system in the uterus has been extensively studied. More recently, it was determined that these same genes are also induced in peripheral immune cells and other tissues throughout the body. In addition to interferon tau and progesterone, pregnancy associate glycoproteins and chaperonin 10 (aka Early Pregnancy Factor) are implicated in altering immune function both locally and systemically during pregnancy. While it is tempting to speculate that this activation of innate immunity is designed to counteract selective immunosuppression, knowledge of the importance of local and systemic immune activation to the success of pregnancy remains incomplete. This area remains fertile ground for developing better approaches to diagnose and treat infertility in domestic farm species and humans alike.

Purification of pregnancy-associated glycoproteins from late-pregnancy Bubalus bubalis placentas and development of a radioimmunoassay for pregnancy diagnosis in water buffalo females.

BACKGROUND: Pregnancy-associated glycoproteins (PAGs) were first described as placental antigens present in the blood serum of the mother soon after implantation. Here, we describe the purification of several pregnancy-associated glycoproteins from water buffalo placenta (wbPAGs). A specific radioimmunoassay (RIA) was developed for early pregnancy diagnosis in buffalo species. RESULTS: Amino-terminal microsequencing of immunoreactive placental proteins allowed the identification of eleven wbPAGs sequences [Swiss-Prot accession numbers: P86369 to P86379]. Three polyclonal antisera (AS#858, AS#859 and AS#860) were raised in rabbits against distinct wbPAG fractions. A new RIA (RIA-860) was developed and used to distinguish between pregnant (n=33) and non-pregnant (n=26) water buffalo females. CONCLUSIONS: Our results confirmed the multiplicity of PAG expression in buffalo placenta. In addition, the RIA-860 system was shown to be sensitive, linear, reproducible, accurate and specific in measuring PAG concentrations in buffalo plasma samples from Day 37 of gestation onwards.

Comparison of commercial ELISA blood tests for early pregnancy detection in dairy cows.

The objective of the present study was to compare two commercially available blood-based pregnancy tests, namely BioPRYN, an ELISA for pregnancy-specific protein B (PSPB), and an ELISA for pregnancy-associated glycoprotein (PAG), for early pregnancy diagnosis in dairy cattle using transrectal ultrasonography as a gold standard. Transrectal ultrasonography was conducted 26-58 days after artificial insemination (AI) in 197 cattle from 19 farms. Concurrently, a blood sample was collected for determination of serum PSPB and PAG. Transrectal palpation was performed approximately 120 days after AI to verify that pregnancy was maintained. For PSPB and PAG, there were no significant differences (P>0.05) in sensitivity (98.0 and 97.8%), specificity (97.1 and 91.2%), positive predictive values (99.3 and 97.8%), negative predictive values (91.9 and 91.2%) and accuracy (97.8 and 96.4%). In conclusion, the two blood pregnancy assays were equally efficacious and were highly accurate (based on transrectal ultrasonography as the gold standard).

Incorporation of a rapid pregnancy-associated glycoprotein ELISA into a CIDR-Ovsynch resynchronization program for a 28 day re-insemination interval.

The objective was to compare two resynchronization programs; one that used a blood-based ELISA for pregnancy-associated glycoproteins (PAG) for pregnancy diagnosis so that non-pregnant cows were re-inseminated at 28 d after first TAI, and another that used transrectal ultrasonography for pregnancy diagnosis so that non-pregnant cows were re-inseminated at 35 d after first TAI. The PAG_resynch cows (n = 103) began CIDR-Ovsynch resynchronization on Day 18 after first TAI (Day 0). On Day 25, the CIDR was removed and pregnancy diagnosis with a PAG ELISA was performed. If a cow was not pregnant on Day 25, she was treated with PGF(2α), treated with GnRH 2 d later (Day 27), and TAI on Day 28. Control cows (n = 99) were observed for estrus until Day 25, when they began an identical CIDR-Ovsynch program with pregnancy diagnosis by transrectal ultrasonography on Day 32. If a cow was not pregnant on Day 32, then she was treated with PGF(2α), treated with GnRH 2 d later (Day 34), and TAI on Day 35. There was no difference in pregnancy per AI (P/AI) for either group at first or second insemination. For cows without pregnancy loss, the interval between first and second (P < 0.001) or second and third (P < 0.016) TAI was shorter for PAG_resynch cows compared with Control cows. The interval between first and second or second and third TAI was not different if pregnancy loss cows were included in the analysis. Plasma progesterone concentrations were similar at PGF(2α) treatment, and plasma estradiol concentrations increased similarly after PGF(2α) treatment for PAG_resynch and Control cows. In conclusion, the 28 d CIDR-Ovsynch resynchronization protocol was comparable to a 35 d CIDR-Ovsynch resynchronization protocol that also included estrus detection. Shortened resynchronization protocols that do not require estrus detection may improve reproductive efficiency in dairy cattle.

Technical note: A rapid enzyme-linked immunosorbent assay blood test for pregnancy in dairy and beef cattle.

The ruminant trophoblast produces pregnancy-associated glycoproteins (PAG) that can be detected in the blood of pregnant animals. The objective was to determine the accuracy of a rapid ELISA PAG-based test for the purpose of pregnancy detection in cattle. Blood was sampled from dairy cattle (539 Holstein cows, 173 Holstein heifers, 73 Guernsey cows, 22 Guernsey heifers, and 12 Jersey heifers) and crossbred beef cattle (145 cows and 46 heifers) that were >or=25 d after insemination (range = 25 to 45 d for dairy and 29 to 56 d for beef). Cattle were examined by ultrasonography for detection of pregnancy within 2 d of blood collection. Whole blood or plasma was incubated in a polystyrene tube coated with a monoclonal PAG antibody for 15 min. The tubes were then washed and subjected to sequential incubations with a biotinylated polyclonal PAG antibody (15 min, followed by wash), a horseradish peroxidase-streptavidin solution (15 min, followed by wash), and a peroxidase substrate. Tubes were visually assessed for color after 15 min (clear solution = PAG negative, not pregnant; blue solution = PAG positive, pregnant). Total assay time was approximately 90 min. The ultrasound examination was used as the standard for pregnancy diagnosis. The sensitivity (99.8 +/- 0.2%), specificity (91.7 +/- 1.4%), and negative predictive value (99.7 +/- 0.3%) for the PAG test used in dairy cattle were similar for different breeds and for cows and heifers. The positive predictive value for the test was greater in dairy heifers than in dairy cows (96.5 +/- 1.4% vs. 90.5 +/- 1.7%, respectively). In beef cattle, the sensitivity (100%), specificity (92.3 +/- 3.0%), positive predictive value (95.0 +/- 2.0%), and negative predictive value (100%) for the PAG test were similar for cows and heifers. The accuracy of the test was not different for dairy and beef cattle. In conclusion, the rapid ELISA pregnancy test based on PAG was highly sensitive and specific for pregnancy detection in dairy and beef cattle.

Field experiences with early pregnancy diagnosis by progesterone-based ELISA in sows.

In four Kenyan pig breeding units the pregnancy diagnosis of sows has been carried out in two groups: Group 1 (n = 1911): the sows were transrectaly pregnancy tested between Days 17-22 post-mating by ultrasound. Sows testing non-pregnant immediately received one dose of 400 IU pregnant mare serum gonadotropin (PMSG) (equine chorion gonadotropin, eCG) and 200 IU human chorion gonadotropin (hCG). On showing signs of oestrous, the animals were subsequently artificially inseminated (AI). Group 2 (n = 1923): sows were pregnancy tested by serum progesterone (P4)-based enzyme-linked immunosorbent assay (ELISA) on Day 17 post-breeding. P4 concentrations were categorized as positive (> 5 ng/ml) or negative (< 5 ng/ml). Sows testing nonpregnant immediately received one dose of 400 IU PMSG and 200 IU hCG by injection, and were subsequently artificially inseminated. The following parameters were evaluated: sows diagnosed non-pregnant, days from first post-weaning insemination until the sows were inseminated at their first return to oestrus; farrowing rate and total piglets born and number of live-born piglets in litters. The percentage of sows diagnosed non-pregnant in the two groups, as well as the totals of born piglets and of live-born piglets in litters did not differ significantly between the two groups. The number of days from the first post-weaning mating until the sows were artificially inseminated at their first return to oestrus and the administration of eCG and hCG was shorter (P < 0.01) and farrowing rate was higher (P< 0.01) in the ELISA-tested sows.

Measurement of EPF for detection of cow pregnancy using rosette inhibition test.

Early embryonic death of calves due to sub-fertility in cows is of great economic concern to dairy industry. Early pregnancy factor (EPF) is a secretory protein with pregnancy associated immunosuppressive properties. Rosette inhibition test (RIT) was used to detect EPF in inseminated dairy cows. Blood samples were collected at two intervals, 1-3 and 5-7 days after insemination from 23 inseminated and 18 non-inseminated control cows for RIT and pregnancy diagnosis performed between 42 and 45 days on palpation. The study indicates that RIT (P<0.05) has the potential to distinguish pregnant from non-pregnant dairy cows in the first week of pregnancy.

Concentration of bovine pregnancy associated glycoprotein in plasma and milk: its application for pregnancy diagnosis in cows.

Pregnancy diagnosis is an important part in reproduction management of ruminants. The aim of the study was to use a new method for evaluating the bPAG and cPAG in milk and blood bPAG and compare this results with the other method for pregnancy diagnosis in the cows. The study was carried out in 220 Holstein Frisian cows. Heparinised blood samples were taken from the jugular vein and stored at -20 degrees C until PAG assay by RIA. For bPAG and cPAG, RIA test, milk samples were homogenized. Pure bPAG was used as a standard tracer described by Zoli et al. (1992). The cows were diagnosed as pregnant by means of USG (Aloka SSD 210) and by rectal palpation. bPAG and cPAG concentration in milk increased after 28 day of pregnancy and showed the rapid increase near the parturition. The same results of bPAG concentration we obtained in the blood samples. The decline of bPAG concentration was faster in the milk than in the blood. The data showed that the RIA method is precise enough to measure PAG concentrations in the maternal blood and milk of cows. The data indicate that milk samples can be used for pregnancy diagnosis in cows. The sensitivity and specificity of RIA measurement of PAG are very high.

Accuracy of a pregnancy-associated glycoprotein ELISA to determine pregnancy status of lactating dairy cows twenty-seven days after timed artificial insemination.

To determine the accuracy of a pregnancy-associated glycoprotein (PAG) ELISA in identifying pregnancy status 27 d after timed artificial insemination (TAI), blood samples were collected from lactating Holstein cows (n = 1,079) 27 d after their first, second, and third postpartum TAI services. Pregnancy diagnosis by transrectal ultrasonography (TU) was performed immediately after blood sample collection, and pregnancy outcomes by TU served as a standard to test the accuracy of the PAG ELISA. Pregnancy outcomes based on the PAG ELISA and TU that agreed were considered correct, whereas the pregnancy status of cows in which pregnancy outcomes between PAG and TU disagreed were reassessed by TU 5 d later. The accuracy of pregnancy diagnosis was less than expected when using TU 27 d after TAI (93.7 to 97.8%), especially when pregnancy outcomes were based on visualization of chorioallantoic fluid and a corpus luteum but when an embryo was not visualized. The accuracy of PAG ELISA outcomes 27 d after TAI was 93.7, 95.4, and 96.2% for first, second, and third postpartum TAI services, respectively. Statistical agreement (kappa) between TU and the PAG ELISA 27 d after TAI was 0.87 to 0.90. Pregnancy outcomes based on the PAG ELISA had a high negative predictive value, indicating that the probability of incorrectly administering PGF(2alpha) to pregnant cows would be low if this test were implemented on a commercial dairy.

Evaluation of early conception factor lateral flow test to determine nonpregnancy in dairy cattle.

The early conception factor (ECF) lateral flow test was evaluated for its ability to accurately determine nonpregnant status in dairy cattle. Results of 2 field trials involving 191 cows and 832 tests indicated the probability that a cow can be correctly diagnosed as nonpregnant by using the ECF test is only about 50%. Agreement of test results between milk and serum obtained from the same cow was 57.5%. The ECF test was not consistent in identifying nonpregnancy when the same cows were tested repeatedly over a period of 4 weeks. We conclude that the ECF lateral flow test does not accurately identify nonpregnancy in dairy cattle.

[Relaxin: an important indicator of canine pregnancy]. / Relaxin: ein wichtiger Graviditätsindikator bei der Hündin.

In the present study the concentration of relaxin in peripheral blood plasma was assessed during canine pregnancy for its suitability as a pregnancy indicator, using a newly developed relaxin enzyme immunoassay. A significant relaxin increase was found in pregnancy at day 24 after ovulation. However, this relaxin increase did not correlate either with litter size or with body weight of the bitch. Induction of abortion with prostaglandin F2 alpha resulted in reduced peripheral relaxin levels, suggesting a damage of the placenta due to this medical intervention. Thus, the results confirm that relaxin, which is produced by the placenta, is a useful marker for early pregnancy diagnosis in the bitch. Relaxin measurement is recommended for detection of pregnancy either alone, or as supplement of ultrasonographic findings.

Early pregnancy diagnosis in swine by direct radioimmunoassay for progesterone in blood spotted on filter paper.

A direct radioimmunoassay method for the measurement of progesterone in blood dried on filter paper has been developed for the early pregnancy diagnosis in sows, as well as for monitoring progesterone levels during the oestrous cycle. Pregnancy diagnosis was performed with 95 sows on Days 17-22 after artificial insemination (AI). The cut-off value for pregnancy diagnosis of 7.5 ng/ml was calculated (mean+/-2S.D.) from the progesterone concentrations measured on the same days from non-inseminated animals. There were 85 cases considered pregnant on the basis of progesterone concentration, leaving 10 animals non-pregnant. The accuracy for the positive cases was 98.8%. Two of the 10 sows considered as negative subsequently farrowed, giving an accuracy of 80%. The overall accuracy of the method was 96.8%. The blood-spot assay may be a useful tool for early pregnancy diagnosis in swine, with respect to sampling, simplicity, speed and accuracy.

Evaluation of the early conception factor (ECF) test for the detection of nonpregnancy in dairy cattle.

The ability to detect conception and/or conception failure in cattle would be beneficial to producers in formulating reproductive management plans. A new diagnostic test, the early conception factor (ECF) test, has been developed forthis application yetthe accuracy of this test has not been adequately determined. The objectives of this study were to evaluate the effectiveness of the ECF test for detecting the nonpregnant cow, and to compare the reliability of serum versus milk ECF tests relative to actual pregnancy rates. In Trial 1, Holstein heifers were synchronized, the animals were bred (timed-AI), and serum ECF tests were performed 72 h later. Heifers exhibiting a negative ECF test after AI were re-synchronized, bred again, and re-tested for ECF for up to three services. Relative to actual pregnancy rates, a negative ECF test was correct (i.e., true negative) 38.5% of the time over the three services. In Trial II, Holstein heifers were bred (AI) after observed estrus and serum ECF tests conducted between Days 1 and 3 and Days 7 and 9 after AI. In this trial, only 44.4% and 55.6% of the confirmed nonpregnant heifers were identified correctly by serum ECF analysis at Days 1 to 3 and Days 7 to 9 post-AI respectively. In Trial III, 40 lactating cows were synchronized, the animals were bred (AI), and serum and milk ECF tests were performed on Days 3, 9, 15, 21 and 30 after AI. Pregnancy diagnosis (ultrasound on Day 30 and palpation on Day 51) confirmed that 50% of the cows were pregnant to AI, while serum and milk ECF analysis indicated a 100% and 37.5% predicted pregnancy rate, respectively, at 30 d post-AI. Moreover, results of the serum and milk ECF tests disagreed with one another 36.9% of the time overall, while agreement between ECF and actual pregnancy rates were 50.6% and 45.6% for milk and serum respectively. Additionally in Trial III, a negative ECF result only identified 5% and 28.8% of nonpregnant cows overall for serum and milk tests respectively (i.e., true negatives), with a high incidence of false positive ECF results noted (47.5% and 31.3% for serum and milk, respectively). Collectively, these data indicate that the current ECF test cannot accurately identify the nonpregnant cow with the precision needed by the dairy producer.

Pregnancy-associated glycoproteins (PAG) detection in milk samples for pregnancy diagnosis in dairy goats.

In ruminants, glycoproteins synthesized in the outer layers of the trophoblast enter the maternal bloodstream. The assay of their concentration in plasma or in serum can be used for pregnancy diagnosis and to indicate feto-placental health status. In this study, concentrations of the pregnancy-associated glycoprotein (PAG) were investigated in the milk of dairy goats. The mean concentrations of PAG in milk were significantly higher in pregnant than in non-pregnant goats throughout the sampling period. Positive milk PAG levels were found in all pregnant animals from Day 32 after breeding, but remained at negative values in nonpregnant ones. This study reports for the first time a pregnancy diagnosis based on milk concentrations of PAG, that could be of great use on the farm.

Noninstrumented enzyme-linked immunosorbant assay for detection of early pregnancy in macaques.

A practical, noninstrumented enzyme-linked immunosorbant assay (NELISA) for the measurement of urinary monkey chorionic gonadotropin (mCG) has been developed for the detection of early pregnancy in macaque monkeys for use in both the laboratory and the field. Five rhesus monkeys (Macaca mulatta) and six crab-eating monkeys (Macaca fascicularis) were tested for the presence of mCG in urine on gestational days (GDs) 12 to 35. The mCG NELISA detected pregnancy as early as GD 14, with an average earliest detection at GD 16.5 +/- 1.4 (n = 11). Out of 90 tests, 27 false-negative and zero false-positive tests were obtained, for an accuracy of 70.0%. Without the aid of a spectrophotometer, the presence of mCG in pregnant monkey samples was indicated by a dark green color change. Nonpregnant monkey urine samples, on the other hand, exhibited no color change. These findings suggest that the simple, economical, and reliable urinary mCG NELISA may be useful for diagnosing early pregnancy in these and related species. Because capture and restraint are unnecessary for collecting urine samples, the mCG NELISA has widespread potential for confined and free-ranging animals.

Radioimmunoassay of a bovine pregnancy-associated glycoprotein in serum: its application for pregnancy diagnosis.

A sensitive and specific double-antibody RIA for a bovine pregnancy-associated glycoprotein (bPAG) is described. The limit of detection was 0.2 ng/ml. The assay was specific for bPAG in that pituitary and placental gonadotropic hormones and other placental or serum proteins assayed in serial dilutions did not cross-react. The RIA allowed measurement of bPAG in placental extracts, fetal serum, fetal fluids, and serum or plasma of pregnant cows. About 20% of unbred heifers and nonpregnant cows had detectable levels ranging from 0.30 +/- 0.09 to 0.50 +/- 0.17 ng/ml (mean +/- SD), and 15% of bull sera showed higher concentrations (3.01 +/- 1.73 ng/ml) of bPAG or bPAG-like protein. Variations among animals was observed in fetal serum bPAG concentrations. Bovine PAG was detected in maternal peripheral blood at Day 22 of pregnancy (mean +/- SD, 0.38 +/- 0.13 ng/ml) in some animals and at Day 30 in all pregnant cows. Peripheral serum bPAG levels increased progressively to 3.60 +/- 1.73 ng/ml (mean +/- SD) at Day 30 of pregnancy, to 24.53 +/- 8.81 ng/ml at Day 120, and to 1551.91 +/- 589.68 ng/ml at Day 270. Peak concentration of bPAG was 2462.42 +/- 1017.88 ng/ml and it occurred 1-5 days prior to parturition. After delivery, bPAG concentrations decreased steadily to 499.63 +/- 267.20 ng/ml at Day 14 postpartum (pp), 10.12 +/- 7.84 ng/ml at Day 60 pp, and 1.44 +/- 1.08 ng/ml at Day 90 pp. The undetectable concentration (less than 0.20 ng/ml) was reached by Day 100 +/- 20 pp. An investigation undertaken in Holstein heifers, Holstein cows, and Hereford cows used as recipients for purebred Holstein embryos supplied evidence of the influence of breed of recipient and sex of fetuses on peripheral concentrations of bPAG. A herd of 430 Holstein-Friesian heifers that had received transferred embryos were bled at Day 35 postestrus (pe) for measurement of bPAG. The bPAG was detected in 287 of 430 serum samples analyzed. By rectal palpation performed at Day 45 pe, 267 heifers with detectable levels of bPAG at Day 35 pe were confirmed to be pregnant as were 3 of 143 heifers previously diagnosed as not pregnant by RIA. These results suggest that detection of this placental-specific antigen in the serum could be used as a specific serological method for early pregnancy diagnosis in cattle from 28 days after breeding.

An immunochemical demonstration of a pregnancy-specific protein in the horse and its use in the serological detection of early pregnancy.

Two-dimensional crossed immunoelectrophoresis of sera from pregnant and non-pregnant horses, using antisera developed against early pregnant mare serum, revealed the presence of two immunologically related proteins one of which appeared to be specific to the pregnant state. This pregnancy-specific protein had beta 2-electrophoretic mobility and was first detectable at Day 6 after successful mating with a stallion. The second protein had gamma 2-electrophoretic mobility and was present in sera from pregnant and non-pregnant horses. The proteins were termed beta 2-horse pregnancy protein and gamma 2-horse protein respectively. The latter appeared to be immunologically related to the former in that the precipitin lines of the 2 proteins showed continuity. Samples from 16 mares mated with a stallion were investigated for the beta 2-protein during the first 3 weeks after mating. Of the 11 successful matings, confirmed by ultrasonic scanning at 90 days or by a successful outcome, 10 mares showed the presence of the protein. In all of 14 non-pregnant sera taken from mares not recently mated, the protein was not detectable. The validity of detection of beta 2-protein as an indication of pregnancy was clinically significant at the 10% level. The presence of the protein in 2 out of the 5 recently mated mares that did not become pregnant may be indicative of a biochemical pregnancy that failed at a later stage of gestation.

[Reliability of pregnancy diagnosis in sows using direct radioimmunoassay of estrone-sulfate]. / Spolehlivost diagnostiky gravidity u prasnic prímou radioimunoanalýzou estron-sulfátu.

The pregnancy diagnosis in sows using direct radioimmunoassay of estrone-sulphate in the blood serum without sample extraction is described. It was found that for the pregnancy diagnosis the period between days 22 and 30 of pregnancy can be used since in this period the estrone-sulphate concentrations in all pregnant sows markedly exceeded those of 64 non-pregnant animals. The estrone-sulphate estimation cannot be used for the pregnancy diagnosis before day 22 and between day 30 and 40 because the estrone-sulphate concentrations in most samples collected in these periods were lower than 4 nmol.l-1.

Assessment of pregnancy in sheep by analysis of plasma progesterone using an amplified enzyme immunoassay technique.

An amplified enzyme immunoassay kit for progesterone analysis was used to diagnose pregnancy in a flock of 130 mule ewes. An accuracy of 100 per cent was obtained after the analysis of progesterone in plasma samples taken 15 to 16 days after mating. In mule ewes a plasma progesterone level greater than 5.9 nmol/litre was indicative of pregnancy. In the validation of the technique, duplicate ewe plasma samples and progesterone standards were compared with a radio immunoassay technique; the regression coefficient between the two techniques was r = 0.82.