Measurement and clinical significance of equine fetal protein in pregnant mares serum

Refereed

MEASUREMENTAND CLINICALSIGNIFICANCEOF EQUINE FETAL PROTEININ PREGNANTMARESSERUM Keld Sorensen, PhD1; Dean P. Neely VMD, PhD2; William Read3; and Paul M. Grappell, MD~

ABSTRACT

An assay for the determination of the concentration of an equine fetal protein (EQFP) in pregnant mares serum was developed and used to quantitate more than 2000 serum samples from Thoroughbred mares during pregnancy, The concentration of EQFP increased throughout pregnancy. Mares with unproblematic (normal) pregnancies were used to establish the reference values at given gestational ages (GA). Mares with pregnancy failures (n=58) were found to have EQFP concentrations outside the reference values established at given GA's; 10 were high and 48 were low. Twin pregnancies (n=21), placentitis and impending abortion (n=71) were associated with elevated concentration. Twin pregnancies were detected by elevated EQFP concentration, in one case as early as 12 days after breeding.

INTRODUCTION

Alpha-fetoprotein (AFP) is a glycoprotein initially found in the yolk sac and later synthesized by the fetal liver of most mammalian species. Consequently, fetal serum has very high levels of AFP. In humans, elevated maternal serum AFP levels are associated with twins, and numerous adverse conditions and outcomes of pregnancy, including fetal anencephaly, neural tube defects and placental abnormalities. 1° Authors' addresses: IEquiChem Research Institute Ltd, 393 Old Country Rd, Suite 200, Carle Place, NY 11514, Ph, 516-333-0422, FAX 516-3385798. 2MidAt~anticEquine Center, PO Box 188, Ringoes, NJ 08551; 3Happy Valley Farm, 4076 NW 95th Ave. Rd, Ocala, FL 32675.

Volume 10, Number6, 1990

Adverse conditions which may occur during pregnancy in the horse include twin pregnancies, a condition occurring at a rate of about 1:6 to 1:10 in the Thoroughbred mare.4 With ultrasonography, the equine practitioner can detect multiple embryonic vesicles by 12-14 days of gestation.* The sonographic echos provide visualization of the embryonic yolk sac during early gestation. 5In cases where the equine practitioner observes two embryonic vesicles, one can be mechanically ablated to exclude thepotential for twin pregnancy. 1~,4 After 45-60 days of gestation, it becomes more difficult to reliably determine whether the mare is pregnant with twins by rectal linear ultrasound methods. 5 Serum estrone sulfate has been used as a biochemical test for equine pregnancy with concentrations increasing between 34 and 45 days of gestation7 Estrone sulfate concentrations decline rapidly in the mares urine after fetal loss (day 47), but serum estrone sulfate concentrations decline less definitely.6 Neural tube defects (the primary reason for maternal serum AFP screening in humans) are uncommon in equine pregnancies, l°.u but the pregnant mare is prone to uterine and vaginal infections, which may involve the placenta?,2 Current testing for detection of placental infections or other placental anomalies are lacking. 1,5 We developed an equine fetal protein (EQFP) assay to assist in clinical diagnostics of equine gestation. The immunochemical assay for the measurement of EQFP was used to determine whether there was a relations hip between twinning and other adverse fetal conditions and the levels of EQFP in the mare. Our report is the first to describe the scientific utility and practical application of measurements of EQFP in pregnant mares.

417

NUMBER

NUMBER OF SAMPLES PER DAY OF GESTATION OF SAMPLES

Table 1. Inter-assay and intra,assay variations as determined by 6 Quality Control samples on 20 assays.

;15

,~

20 -

15

oooo

:

~%a._ o o

o

--

I

10

,, I

20

Intra-assay CV%

QC-1 QC-2 QC-3 QC-4 QC-5 QC-6

100.6 61.5 41.3 19.4 10.1 5.0

2.80 3.41 3.66 3.91 3.54 3.64

I

I

I

I

I

30

40

80

60

70

.

I

I

80

90

9.06 7.16 6.35 6.02 6.50 9.86

The inter-assay variation (CV%) was determined by the measured values in 20 consecutive assays (duplicate determinations of quality control samples). The intra-assay variation was determined by the mean CV% value measured on the duplication of the Quality Control samples on the same 20 assays. The assays were run on 20 days, with 2 different lot

5

0

Value Interassay units/ml CV%

,., @Z

10

5

Sample

100

GESTATIONAL AGE (DAYS) Figure 1. The number of samples at given days of GA. The median number of samples drawn per mare was 3.

MATERIALS AND M E T H O D S

Serum samples Serum samples (2053) were collected from 522 mares atbreeding farms in Florida, Kentucky and New Jersey. The median number of samples per mare was 3. The serum was mailed to our laboratory, and was analyzed on the day of arrival. Each sample was accompanied by a clinical data form, containing pertinent information on ultrasound observations, results of palpation, medical history, medication given, etc. ELISA for EQFP

A microtiter plate enzyme linked immunosorbent assay was used (ELISA) for the determination of EQFP. The assay is based on polyclonal and monoclonal antibodies, and is an in-house development of EquiChem Research Institute. Quality assurance: All samples were tested in duplicate. All microtiter plate assays have 6 quality control samples (3 at the beginning of the assay, 3 at the end), and 7 calibrators, all of which also were tested in duplicate. The quality control samples consist of serum samples spiked with purified EQFP. The calibrators consist of purified EQFP in a matrix devoid of EQFP. In order to facilitate comparison of laboratory results on individual mares at any given GA, actual measured IU/ ml values were converted to SD-units (called SDR - standard deviation ratio). This unit is a direct measure of the degree to which any sample deviates from 'usual' values, independent of parameters such as GA, 3 because the SDR is defined as the measured value minus the established population mean divided by the established population SD at a given GA. 3 418

RESULTS

Figure I gives the number of samples per day of GA. For early pregnancy (GA < 100 days) more than 100 samples per gestational week were collected. Between day 12 and day 60 of gestation, greater than 15 samples per day of GA were obtained. For the later gestational ages, fewer samples were collected (less than 5 per day of gestation). The mean EQFP levels are depicted in Figure2, for the entire gestational length. Actual means are shown as circles (only every 5th day is shown), the smoothed curve, is also shown. For each day of GA <100 days, the mean EQFP value was established as well as the Standard Deviation (SD). at later GA's the value was established for dates with more than 5 samples. The means and SD's were calculated based only on samples from normal pregnancies, i.e. pregnancies where there was no indication of any of the problems believed to be potentially associated with elevated levels of EQFP at the time of blood drawing. The procedure of unifying the data set by the use of SDR units was used throughout the study. The validity of this approach is exemplified in Figure 3, where the distribution curve of all samples in the study is established using the SDR value of each sample. The advantage to using SDR rather than the measured IU/ml was that the interpretation of results on individual mares was facilitated since only one reference range was needed instead of 340 (1 for each GA). Statistical evaluation of groups of mares was performed to analyze concentrations of EQFP (expressed as SDR) for certain conditions. Three groups were analyzed. 1) Unaffected, i.e. pregnancies where the fetus is presumed healthy, without twins, placentitis or other gestational anomaly; 2) Confirmed twinning, diagnosed by serial ultrasonic examinations; 3) Problems, i.e. mares with gestational conditions, such as placentitis, placental abnormalities, embryonic resorption, etc. The levels associated with each of these conditions are depicted in Figure 4. Twins (n=21) have an elevated concentration, with a mean concentration of 2.42 SDR, and the problem (n=71) mares had EQUINE VETERINARYSCIENCE

DISTRIBUTION CURVE OF STUDY SAMPLES

EQFP MEDIAN LEVELS AS A FUNCTION OF GESTATIONAL AGE

NUMBER OF SAMPLES

EQPP CONCENTRATION(UNITS/ml)

i o

0

60

I00

160

200

j

260

-2.4-2.0-L.6-13-.S

300

GESTATIONAL AGE (DAYS) Figure 2. Median EQFP concentrations in lU/ml for gestational ages up to 340 days. The line is the moving average of the actual median values. For reasons of pending patent applications, the actual IU/ml values are not displayed on the y-axis.

EQFP VALUES IN DIFFERENT CATEGORIES OF MARES

-.4

0

.4

.8

1.~ 1.6 3.0 2.4 2.8

),&0

CONCENTRATIONS OF EQFP (SDR) Figure 3. Distribution curve for all samples. Each bar shows the n umber of samples with the EQFP concentration shown at the bottom of each bar. Classwidth .4 SDR. The total number of samples: n=2035.

6

"TWIN" PREGNANCIES EQFP CONCENTRATION(SDR)

EQFP CONCENTRATION(SDR)

6, i

---

4'

21 _~

0

1

I

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i

2o

40

6o

80

too

~o

GESTATIONAL AGE (DAY)

-11

--e-A

--Z-B --~-C

-*--D

-,X-E

-2 NORMAL

CONCEP. FAILURES

TWINS

PLACENTITIS

EMBRYONIC LOSS

Figure 4. EQFP concentrations (Mean _+SD) for normal pregnant mares, mares confirmed by serial sonographical exam to be carrying twins (n=21), mares with placentitis, placental abnormalities and fetal distress (n=71) as well as mares with failure of conception (n=10) and mares with early embryonic loss (n=48). For this figure, the group of early pregnancy failures (n=58) was divided into failure of conception and early embryonic loss based on the EQFP levels. This is an operational definition.

even higher values, with a mean of 3.82 SDR. Mares (n=58) who either failed to conceive or had early embryonic loss were also investigated (no attempt was made to distinguish these two groups), The samples from such mares were analyzed, and found to fall in two different groups, namely with high EQFP values and with low EQFP values. A total of 10 mares showed high values, with an

V o l u m e 10, N u m b e r 6, 1 9 9 0

Figure 5. This graph demonstrates EQFP values of mares who were initially believed to be pregnant with twins, based on sonographical information. The mares with high values were confirmed on repeat sonographical examination, the mares with low values could not be confirmed as carrying twins.

average concentration of 2.99 SDR (range 1.8 - 5.7 SDR). The gestational age was between day 12 and 35; mean GA 22.2 days. The remainder of the samples from mares where the conception apparently failed, had a mean level of-1.1 SDR (range -2.3 - 0.0 SDR). For both groups of pregnancy failure, the Mean + SD is depicted in Figure 4.

Case study mares Case studies were selected based on their individual clinical conditions. Some mares were not diagnosed as having problems until the result of the EQFP test was known.

419

detected after a sudden rise in EQFP values. EQFP concentrations had previously been normal, but reached very high levels during progression of the infectious placentitis. The placentitis was subsequently confirmed microbiologically. Mare G (Fig. 6) was diagnosed by sonographical examination as undergoing a fetal death. This was reflected by increased EQFP values at the time of the fetal death near day 65-75 GA.

EQFP CONCENTRATIONS IN CASES OF COMPLICATIONS 8

EQFP CONCENTRATION (SDR) '

,

~1

-

'"

DISCUSSION 0 I

0

l

I

I

l

I

10

:30

30

40

60

| ....

60

J

I

L

70

80

90

|00

GESTATIONAL AGE (DAYS)LOSS --,0- MARE F --"- MARE G

Figure 6. Two advanced pregnancies with elevated EQFP. One mare was shown to have a placentitis (circles), the other had fetal death confirmed sonographicatly between

day 65 and 75 (squares).

Mare A (Fig. 5). A sample pre- and immediately postreduction of sonographically confirmed twin pregnancy was obtained. Both levels were high. A later EQFP sample indicated successful ablation (low value). Mare B (Fig. 5) carried sonographically confirmed twins during the first 3 samplings. Reduction of one twin was performed. The EQFP concentration did not return to normal values. In the past several breeding seasons, the mare has aborted (both singleton and twins), due to an abnormal placenta. Sonographic examination of the placenta was unsuccessful in demonstrating any anomaly, due to instrument limitations. Histological evidence of the type of placental anomaly determined in the past, was not available. It is possible that the late ablation (day 45) resulted in embryonic remaining placentation or endometrial cups, that maintained the elevated EQFP. Mare C (Fig. 5) had high EQFP concentrations, which correlated with sonographically conftrmed twins. The owner of the mare wanted pregnancy to continue without to ablate one embryo. The EQFP concentrations remained high during the study. Mare D (Fig. 5) was diagnosed sonographically as suspect twins, however EQFP values were low. Without knowledge oftheEQFPresult, the sonographical exam was repeated 7 days later, and revealed that the mare was open, i. e. no longer pregnant. Mare E (Fig. 5) sonographically was believed to carry twins, howeverEQFP values were low. Withoutknowledge of the EQFP result, the sonographical examination was repeated 7 days later, and reported by the attending veterinarian as a blighted twin. A possible cause for misinterpretation of twins by ultrasound scan would be the presence of uterine cysts of similar size to an embryonic vesicle. Mare F (Fig. 6) had an advanced pregnancy with severe vaginal discharge associated with a bacterial infection 420

This paper describes for the first time that pregnant mares have measurable levels of EQFP in their serum which can correlate with viable fetal parameters and the clinical status of the mare. It can be speculated that the underlying biological reason for our findings can be found in the leakage of fetal material into the mare. In the case of twin pregnancy, the double embryonic/fetal mass will be reflected in higher mare EQFP levels. The group of mares where pregnancy failed, fell into two groups (low and high values). It can be theorized, that low values were obtained from mares with failures of conception and high values from mares with embryonic loss and resorption of fetal material. Our study was not designed to clinically distinguish the two groups. It is expected that this biochemical test will be a valuable adjunct to practicing veterinarians in the following ways: •Biochemical indication of the presence of twins, to support sonographic findings. •Biochemical confmnation of successful ablation of one twin conceptus. •Biochemical indication of placentitis. •Biochemical indication of fetal viability or fetal compromise. •Biochemical indication of probable impending abortion.

REFERENCES

1. BallBA, Woods GL: Pregnancywastagein mares. Soc for Therio, Proc Annl Mtg 120-125,1986. 2. BallBA, Woods GL: Embryonicloss and early pregnancy loss inthe mare. Comp ContEduc 9:459-470,1987. 3. In: Clinical Chemistry: Theory, analysis and correlation. LA Kaplan, AJ Pesce, eds. CV Mosby, St. Louis, MO, 1984. 4. GintherOJ :Twinning in mares: a reviewof recent studies. Equine Vet Sci 127-135,1982. 5. GintherOJ: Embryonic loss in mares: incidence, time of occurrence and hormonal involvement. Therio 23:77-89,1985. 6. KasmanLH, Hughes JP, Stabenfeldt GH, Startt MD, Lasley BL: Estrone sulfate concentrations as an indicator of fetal demise in horses. Am J VetRes 49:184-187,1988. 7. KindahlH, Knudsen O, Madej A, Edquist LE: Progesterone, prostaglandinF-2a, PMSG and estrone sulphateduring early pregnancy in the mare. J Reprod Fert Suppl 32:353-359,1982.

EQUINE VETERINARY SCIENCE

8. MacriJN, Haddow JE, Weiss RR: Screening for neural tube defects in the United States. Amer J Obstet Gynecol 133:119,1979. 9. Pascoe DR, Pascoe RR, Hughes JP, Stabenfeldt GH, Kindahl H: Managementof twin conceptuses by manualembryonic reduction: comparison of two techniques and three hormone treatments. Am J Vet Res 48:1594-1599. 10. van Niekerk CH, Allen WR: Eadyembryonic development in the horse. J Reprod Fort (Suppl) 23:495-498,1975. 11. Whitwell KE: Investigationinto fetal and neonatal losses in the horse. Vet Clin NAmer: Lg Anita Pract 2:313-331,1980.

ERRATA There is an error in Table 1 of the article Chelatedmineralsupplementation in the barren mare: A preliminary trial, Equine Veterinary Science, 1990, 10:176- 181. The term Est. Net Energyd,~(Mcal/kg) should be replaced by the term DigestibleEnergy(Mcal/kg) d,',The corresponding energy values should be: Control Chelate Inorganic Pasture NRC Digestible Energy (Mcal/kg)

3.2

3.2

3.2

2.1

2.0

The e superscript from the legend should read Digestible

Energy(Mcal/kg)= % total digestible nutrients X 0.0444.

We regret that this error occurred.

ERRATA The following corrections should be made to the article Comparative study of muscle fiber type composition in the middle gluteal muscle of Andalusian, Thoroughbred and Arabian horses, Equine Veterinary Science Vo[ 9. No 6. Page338, line 9, reference nutuber33 shouldrefer to: Valberg S, Ess~n-Gustavsson B, Skoulund-Walberg H: Oxidative capacity of skeletal muscle fibre types in racehorses: histochemical versus biochemical analysis. Equine VetJ 20:291 295, 1988. Page 338, the second sentence of the RESULTS paragraph should say:"...oftype 1and type2 fibers: the percentageof type 1 was 29.5 in Andalusian, 23.8 in Thoroughbred and 35.2 in Arabianhorses." The editors regret this error. The corrections

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