Early conception factor lateral flow assays for pregnancy in the mare

The ECF™ lateral flow assay test is marketed to detect non-pregnancy in mares. The objectives of the present study were to determine the accuracy of the ECF test, the accuracy of the electronic reader accompanying the ECF test, and agreement between two human readers and the electronic reader. Serum samples were collected from anestrus, cycling but not inseminated, and inseminated mares, and were evaluated with the ECF™ test (EDP Biotech Company, Knoxville, TN, USA) at The Ohio State University and at the EDP Biotech Laboratory. Specificity ranged from 0.07 to 0.16, the negative predictive value ranged from 0.15 to 0.33, and accuracy ranged from 0.43 to 0.52. The electronic reader did not add improve the accuracy or predictive values of the test. Based on the electronic reader, 80.0% of the serum samples collected from the anestrus mares were false positives; Readers 1 and 2 had 60.0 and 33.3% false positives, respectively. For samples collected during the estrous cycle, 83.9% were false positives by the electronic reader, whereas Readers 1 and 2 had 43.7 and 26.4% false positives. We concluded that, regardless of whether the test strips were evaluated by a human or electronic reader, this assay was not accurate for determination of the non-pregnant mare.     

Early growth retardation in diabetic pregnancy.

Thirty-five insulin-dependent diabetic women with reliable menstrual histories were examined by ultrasonic scanning in the 7th-14th weeks of pregnancy. Judged from crown-to-rump length the fetuses were on average 5.4 days smaller than those in a local normal series. Ten of the fetuses were nine days or more smaller than normal and had a lower mean birth weight than the others, though the mean gestational age was similar. Maternal diabetes was not more severe in this group, but only two of the mothers had attended a special hospital for control of their disease as compared with 19 of the remainder. Although babies of diabetic mothers are often overweight, there appears to be a subgroup of cases in which fetal growth is retarded early in pregnancy, leading to low birth weight and possibly a higher incidence of congenital malformations.     

Early pregnancy assessment with transvaginal ultrasound scanning.

OBJECTIVE: To establish normal parameters in early pregnancy through transvaginal ultrasonography so that gestational age can be determined and to correlate the sonographic findings with serum human chorionic gonadotropin (hCG) levels calibrated against the first international reference preparation standard. SETTING: Infertility clinic. PATIENTS: Thirty-five women with normal intrauterine pregnancy. INTERVENTIONS: Serial measurement of the serum hCG level and the diameter of the gestational sac through transvaginal ultrasonography. MAIN RESULTS: The gestational sac could not be visualized when the hCG level was less than 1100 IU/L. The average growth rate of the sac was 0.9 mm/d. The threshold values for sac diameter, serum hCG level and gestational age below which the yolk sac was not visible were 3.7 mm, 1900 IU/L and 36 days respectively; the corresponding values above which the yolk sac was always visible were 6.7 mm, 5800 IU/L and 40 days. The threshold values below which cardiac activity was not visible were 8.3 mm, 9200 IU/L and 41 days respectively, and the corresponding values above which cardiac activity was always visible were 14.0 mm, 24,000 IU/L and 46 days. The mean gestational ages and the 95% confidence and prediction intervals were tabulated so that measurement of the gestational sac diameter could be used to estimate gestational age early in normal pregnancy. CONCLUSIONS: Transvaginal ultrasonography enables detection of an intrauterine sac and reliable estimation of gestational age on the basis of sac dimensions before an embryo can be seen.     

Early pregnancy azathioprine use and pregnancy outcomes

BACKGROUND: Azathioprine (AZA) is used during pregnancy by women with inflammatory bowel disease (IBD), other autoimmune disorders, malignancy, and organ transplantation. Previous studies have demonstrated potential risks. METHODS: The Swedish Medical Birth Register was used to identify 476 women who reported the use of AZA in early pregnancy. The effect of AZA exposure on pregnancy outcomes was studied after adjustment for maternal characteristics that could act as confounders. RESULTS: The most common indication for AZA use was IBD. The rate of congenital malformations was 6.2% in the AZA group and 4.7% among all infants born (adjusted OR: 1.41, 95% CI: 0.98–2.04). An association between early pregnancy AZA exposure and ventricular/atrial septal defects was found (adjusted OR: 3.18, 95% CI: 1.45–6.04). Exposed infants were also more likely to be preterm, to weigh <2500 gm, and to be small for gestational age compared to all infants born. This effect remained for preterm birth and low birth weight when infants of women with IBD but without AZA exposure were used as a comparison group. A trend toward an increased risk of congenital malformations was found among infants of women with IBD using AZA compared to women with IBD not using AZA (adjusted OR: 1.42, 95% CI: 0.93–2.18). CONCLUSIONS: Infants exposed to AZA in early pregnancy may be at a moderately increased risk of congenital malformations, specifically ventricular/atrial septal defects. There is also an increased risk of growth restriction and preterm delivery. These associations may be confounded by the severity of maternal illness. Birth Defects Research (Part A), 2009. © 2009 Wiley‐Liss, Inc.     

Atrial natriuretic factor in pregnancy and pregnancy-induced hypertension.

In normal pregnancy, cross-sectional clinical data do not consistently show plasma ANF concentration differences between early pregnancy and the nonpregnant state. Sequential data in the baboon (but not in rat) show a significant decrease in plasma ANF concentration and in cardiac filling pressures in early pregnancy. The latter data support the view that pregnancy is an underfill state secondary to a primary vasodilatation. Cross-sectional and longitudinal studies in normal pregnancy in humans show that plasma ANF levels tend rise to values that are, in the third trimester, higher than in the nonpregnant state. However, late postpartum sequential data (1.5-3 months) in humans do now show a significant drop in plasma ANF concentrations, suggesting that plasma ANF is not actually increased in normal pregnancy. In the baboon (but not in the rat) there is a steady rise in plasma ANF levels to values that are significantly higher in third trimester than before pregnancy. These data suggest that in human pregnancy, in contrast with the baboon, the plasma volume expansion induced by normal pregnancy is not sensed as such by the atria probably because of an isopressive adaptation of plasma volume to an enlarged vascular bed. However, acute decrease or increase of venous return induced by low sodium diet, changing position or infusion of isotonic saline are sensed as such by the atria in normal pregnancy as in the nonpregnant state.(ABSTRACT TRUNCATED AT 250 WORDS)     

Relationship between early pregnancy factor, mouse embryo-conditioned medium and platelet-activating factor.

The effects of synthetic platelet-activating factor (PAF-acether) and mouse embryo-conditioned medium (a source of embryo-derived PAF (EPAF)) on production of early pregnancy factor (EPF) were compared. Embryo-conditioned medium, itself inactive in the EPF bioassay, stimulated ovarian production of EPF in vitro but PAF-acether did not. In vivo, embryo-conditioned medium induced EPF activity in serum of oestrous female, but not in male, mice in contrast to PAF-acether, which induced activity in serum of both male and female mice. This PAF-induced activity was transitory, declining significantly by 2 h and disappearing by 3 h after injection. Activity induced by embryo-conditioned medium was first evident at 2 h after injection, serum concentrations increasing up to 6 h after injection. By discriminating between the behaviour of PAF-acether and EPAF, these studies reinforce the conclusions of other workers that the molecule produced by the embryo is not PAF. Further investigations into the mechanism of action of PAF-acether revealed that it is a potent inducer of activity in the EPF bioassay, with an absolute requirement for platelets in the spleen cell suspension used in the assay. This platelet-derived active species was bound specifically by an anti-EPF monoclonal antibody, indicating that it is EPF-like. This is consistent with parallel studies showing that platelets are not required for induction of activity by either pregnancy serum or purified EPF. These studies were applied to the PAF-induced leukotriene-like species, which had been found by others to be active in the EPF bioassay. Pregnancy serum induced the appearance of this substance from the spleen cell suspension used in the assay; thus the leukotriene-like substance may be regarded as an effector molecule in vitro or mediator of the initiating stimulus of EPF in the bioassay.     

Prefertilization treatment of mice with platelet activating factor affects pregnancy.

Embryo-derived platelet activating factor (EPAF) is thought to be either biologically similar to platelet activating factor (PAF) or responsible for PAF liberation in vivo. We have previously shown that premating PAF treatment in the mouse renders the platelets nonresponsive to EPAF, leading to a reduced implantation rate in these animals. In this study, we have shown that females, injected with PAF before mating, show altered embryo development invivo on day 4 postfertilization. This is manifested as an interruption of compaction, a reduced cell number per embryo, and reduced embryo number per mouse. Results suggest that EPAF represents an early pregnancy signal that supports embryo development. The most likely mechanism is via platelet activation, since only those mice that showed thrombocytopenia after fertilization were found to have normal embryos on day 4 postmating.     

Early diagnosis and treatment of ectopic pregnancy

During the period from 1995 to 1999, 64 patients were treated for ectopic pregnancy. All patients admitted to the department passed the same procedure including (complete preoperative laboratory findings, Beta HCG, serum progesterone and transvaginal colour Doppler). In patients who had ultrasound finding typical for ectopic pregnancy in combination with positive Beta HCG, conservative treatment was primarily done. In the rest of the patients, Beta HCG was tested every second day and in combination with the clinical and vaginosonographical findings the patients underwent diagnostic or operative laparoscopy. Out of 64 patients 36 had visible ectopic pregnancy when admitted to the clinical department. Three patients had no visible ectopic pregnancy neither at the time of their admission to the department nor at the time of laparoscopy. One of them had cervical pregnancy and the other two had pregnancies in the uterine part of the tube. The patient with cervical pregnancy was treated with metrotrexat (MTX) 12 mg daily in 5 doses. One patient with cornual pregnancy was treated with high doses of oxytocin infusion in combination with MEB intravenously 3 x 1 amp. The other patient with cornual pregnancy underwent laparoscopy with cornual resection and salpingectomy. Four of other tubar pregnancies were treated with metrotrexat 12 mg/day for 5 days. Other ectopic pregnancies were treated as follows: 36 laparoscopic salpingectomies, 10 laparoscopic salpingotomies with ovum expression, 9 adnexectomies by laparotomy, and 2 laparoscopic adnexectomies.     

Early pregnancy associated thrombocytopenia as an initial response to pregnancy in cattle

This study was conducted to determine if early pregnancy-associated thrombocytopenia exists in cattle as has been demonstrated in mice and in humans. Three experiments were designed to compare peripheral platelet counts in pregnant versus nonpregnant animals. In Experiment 1 heifers (n = 25) were artificially inseminated 12 h after the onset of estrus. Peripheral platelet counts in 19 pregnant versus 6 nonpregnant heifers did not reveal any significant differences between groups after insemination. In Experiment 2 embryos were collected nonsurgically from superovulated cows (n =18) on Days 6 to 7 after estrus. Platelet counts were monitored every 12 h after the first insemination until 60 h after the second insemination. Platelet counts and the number of embryos collected nonsurgically from these superovulated donors did not show any significant correlations (P>0.05). Ten recipient heifers synchronized to donor animals received either an unfertilized ovum or a good quality embryo via nonsurgical transfer into the uterus. There were no significant reductions in platelet counts after transfer. In Experiment 3 platelet counts were monitored daily in four pregnant and five nonpregnant recipient heifers between Day 0 and Day 30 after embryo transfer on Day 8 of the cycle. The platelet counts did not reveal any significant differences between the pregnant and nonpregnant groups throughout Days 0 to 30. These results indicate that early pregnancy-associated thrombocytopenia cannot be demonstrated in cattle. Peripheral platelet counts cannot be used as an indicator of early pregnancy in cattle.     

Early pregnancy termination in rats immunized with denatured chicken riboflavin carrier protein.

Immunoneutralization of the maternal riboflavin carrier protein in the pregnant rat with antibodies to chicken egg vitamin carrier has earlier been shown to terminate their pregnancies. In order to understand the nature of the epitopic conformations capable of eliciting antibodies bioneutralizing the endogenous riboflavin carrier protein in the pregnant rat, we compared pregnancy progression in the fertile rodents following active immunization with either the native, SDS-denatured, reduced-carboxymethylated or SDS-treated reduced carboxymethylated avian egg white riboflavin carrier protein. The data revealed that despite the total antibody titers being higher in the animals immunized with the native protein, the antibodies elicited against the denatured avian vitamin carrier exhibited relatively better potencies to bioneutralize the endogenous maternal protein as evidenced by higher rates of early fetal resorption.     

Early pregnancy diagnosis in the ewe, based on milk progesterone levels.

Plasma and milk progesterone concentrations in pregnant sheep (18--22 days after mating) were similar, about 3.7 ng/ml whereas values in non-pregnant sheep were less than 1 ng/ml. Lambing results indicated identical accuracy for both methods (82 and 84% in 2 flocks). The accuracy was 92--100% for ewes diagnosed non-pregnant in the breeding season, but for ewes tested in the non-breeding season the diagnosis of non-pregnancy according to milk progesterone levels was only 50% accurate.     

Purification and characterization of early pregnancy factor from human pregnancy sera

Early pregnancy factor (EPF) is a pregnancy-associated protein detected in the maternal serum by using the rosette inhibition assay and by evaluating the suppression of adoptive transfer of contact sensitivity. Because of its inhibitory effect on the functional reactivity of immunocompetent cells, EPF is thought to be involved in immunoregulation of the maternal immune system during early pregnancy. EPF was purified six million-fold from the serum of pregnant women between 5 and 12 weeks of gestation. The specific activity of purified EPF was approximately 8 x 10(8) units/mg. The purification scheme involved sequential DEAE-cellulose chromatography, S-Sepharose chromatography, concanavalin A-Sepharose chromatography, heparin-Sepharose chromatography, Mono S fast protein liquid chromatography, and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified protein has an apparent molecular weight of 21,500 as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 28,000 by gel permeation high pressure liquid chromatography. The isoelectric point of purified EPF moiety is 6.5. The biological activity was susceptible to the proteolytic enzyme trypsin, acidic pH conditions, organic solvents, and sodium dodecyl sulfate, but stable to heat treatment at 56 degrees C for 30 min and the reducing agent dithiothreitol. The biological and physicochemical properties of EPF appear to be distinct from other pregnancy-associated and immunoregulatory proteins.     

Serum levels of macrophage colony-stimulating factor in singleton and twin pregnancy.

Macrophage colony-stimulating factor (M-CSF) is present in the placenta and has been implicated in placental growth and development. M-CSF levels in peripheral blood increase significantly with progression of pregnancy in uncomplicated singleton pregnancy. This study investigated whether serum M-CSF levels in singleton pregnancy differed from those in twin pregnancy. A hundred and four pregnant women, of whom fifty-nine were women carrying single fetuses and forty-five were women with twin fetuses, participated in the study. Their average gestational age at entry was 10, 20, 30, and 38 weeks of gestation in singleton pregnancy and 10, 20, 30, and 35 weeks in twin pregnancy. Peripheral blood was collected and serum was separated after centrifugation and stored at - 20 degrees C. M-CSF levels were determined by ELISA. In both groups, the serum levels of M-CSF increased significantly as pregnancy progressed. M-CSF levels were relatively higher in twin pregnancy than in singleton pregnancy at 10, 20, and 30 weeks. At 35 and 38 weeks, the M-CSF levels were significantly higher in twin pregnancy than in singleton pregnancy. Elevation of serum M-CSF supports M-CSF production in the placenta. This elevation in twin pregnancy may be related to increased demand of M-CSF in twin pregnancy.     

Early pregnancy diagnosis by transrectal ultrasonography in ewes

Two studies were performed to evaluate early pregnancy diagnosis by transrectal ultrasonography (TRUS), using a 7.5-MHz transducer in ewes. In the first study, the objectives were to determine the earliest date that a reliable pregnancy diagnosis could be made (percentage of ewes detected pregnant between days 15 and 20 after mating). In the second study, the objective was to confirm the findings of the first study using a randomized controlled trial. In both studies, the ewes were restrained in dorsal recumbency, using a special chute that maintained the pelvis at an angle of 30–35° lower than the head. In the first study, 30 Suffolk ewes were synchronized and maintained with 2 rams for 5 days. Each ewe was subjected to the first TRUS on day 15 after mating and daily thereafter until day 20 (estrus = day 0). Pregnancy was defined as the presence of an embryo or extra-embryonic membranes. The percentage of ewes detected pregnant at days 15, 16, 17, 18, 19 and 20 were 0% (0/30), 26.7% (8/30), 86% (24/30), 90% (27/30), 96.7% (29/30) and 100% (30/30), respectively (P < 0.001). In the second study, 390 TRUS examinations (TRUS-1) were performed on ewes from 10 to 50 days after mating in a breeding program (group mating, hand mating, cervical and intrauterine AI; breeding group; n = 270) or with vasectomized rams (vasectomized group; n = 120). The breeding date and the status of breeding were unknown to the operator. Thirty of these ewes were mated with vasectomized rams and used repetitively four times as the non-pregnant control group. All females had a subsequent TRUS (TRUS-2) between 7 and 30 days after the TRUS-1 examination. The second TRUS was used as the standard test against which the performance of the TRUS-1 was compared. The percentage of ewes correctly diagnosed at day 15 or less, days 16, 17, 18, and 19 in the breeding group were 0% (0/29), 31.3% (5/16), 40% (8/20), 70% (7/10), and 100% (14/14), respectively (P < 0.001). All the diagnoses of ewes more than 20 days following mating in the breeding group, were correctly predicted (n = 181), as well as all ewes from the vasectomized group (n = 120). It could thus be concluded that the earliest pregnancy diagnosis using a 7.5-MHz transducer by transrectal route based on the presence of positive signs of pregnancy is at day 16 and the maximum sensitivity and negative predictive value was reached at day 20 following breeding.     

Early pregnancy diagnosis by transrectal ultrasonography in dairy cattle

The objective of the present study was to determine differences in time of detection of pregnancy between heifers and cows and the interval after insemination at which the maximum sensitivity and negative predictive value of transrectal ultrasonography were obtained. One-thousand-four-hundred transrectal ultrasonographies (TRUS-1; 1,079 in cows and 321 in heifers) were performed using a 5-MHz linear-array transducer. The cattle were randomly assigned to have TRUS performed once between days 24 and 30 (estrus=day 0) in cows or between days 21 and 27 in heifers. Every TRUS diagnosis was subsequently compared with a second TRUS diagnosis (TRUS-2), performed 3-8 days later, after day 30 (range 31-38) for cows and after day 27 (range 28-35) for heifers. The sensitivity and specificity between cows and heifers for the common days of TRUS (from 24 to 27) were compared. In cows, sensitivity increased gradually from 74.5% at day 24 to 100% at day 29 (P<0.01). Specificity increased from days 24-25 and reached a plateau of 96.6% on day 26 (P<0.01). In heifers, sensitivity increased from 50% at day 21 to 100% at day 26 (P<0.01). Specificity increased from 87.5% at day 21 and remained steady at 94% starting on day 23 (P>0.05). The sensitivity for cows and heifers was 89.2 and 96.8%, respectively (P<0.05) and the specificity was 93.0 and 93.4% (P>0.05). In this study, heifers were diagnosed pregnant earlier than cows, and the maximum sensitivity and negative predictive value were obtained 3 days earlier in heifers than cows (days 26 and 29, respectively).