Equine Pregnancy Research Articles

Survival of equine trophoblast transplants in immunodeficient mice

Early equine pregnancy is characterized by unique structures known as endometrial cups. The cups form between days 38-40 of gestation after the chorionic girdle trophoblasts invade the endometrium and differentiate into the endometrial cup trophoblasts. The cup trophoblasts secrete equine Chorionic Gonadotrophin (eCG), which promotes the formation of secondary corpora lutea that maintain progesterone levels until about day 100. In most pregnancies the endometrial cups have a lifespan of 60 to 80 days and are no longer viable after day 120 of gestation. Maternal lymphocytes accumulate in striking concentrations around the endometrial cups during their lifespan, suggesting an immunological mechanism leading to their death. However, the role of the maternal immune system in cup destruction is still unresolved, despite many efforts to determine how the cup trophoblasts die. In this study we transplanted horse chorionic girdle trophoblast cells into immunodeficient mice. We hypothesized that if maternal immune responses are responsible for the death of the endometrial cups, the life-span of the transplanted girdle cells would be extended beyond 60-80 days. Chorionic girdlewas isolated from equine conceptuses recovered non-surgically from mares at day 35 of gestation. Small pieces of girdle were injected subcutaneously on the backs of Severe Combined Immunodeficient (SCID) [n1⁄413] and normal Balb/C [n1⁄44] mice. Recipient mice were bled biweekly and serumwas tested for eCG content as an indirect measure of survival of the trophoblast transplants. Mice

Successful isolation and culture of equine placental cells from failed early pregnancies

Early pregnancy loss (EPL) prior to day 65 occurs in 610% of all equine pregnancies and accounts for over 50% of Thoroughbred pregnancies lost in the UK. Despite the clear influence of this condition on fertility, we still know relatively little about the causes of EPL. We hypothesise that a proportion of cases of EPL, in particular in older mares, arise due to chromosomal defects such as trisomies and more rarely translocations. Progress in identifying genetic cause(s) of EPL has been hampered due to a reported lack of failed conceptus tissue and a method to isolate and culture cells obtained from those conceptuses. The objective of this study was to develop a method to isolate and culture placental cells isolated from failed pregnancies by comparing the viability and culture success rates of placental cells cultured in different media and processed at different time intervals following uterine lavage. All Thoroughbred mares included in the study were housed on studfarms in the Newmarket area, UK. Conceptus material from pregnancies that failed prior to day 65 was isolated by sterile uterine lavage performed by the attending veterinary surgeon. Conceptuses were transported to the laboratory on ice in HBSS, 5% Equine Serum, 2.5 mg/ml amphotericin, 20 ng/ml kanamycin and 100 U/ml penicillin-streptomycin. Tissue was isolated from the allantochorion, chorion and chorionic girdle (if present) and cells cultured in three different media; AmnioChrome II,

The effect of equine Chorionic Gonadotrophin (eCG) on luteal vascularity in the mare

Although progesterone is required to maintain the pregnant state, oestrogen is also a feature of early equine pregnancy. As early as day 40 a marked increase in serum oestrogen concentrations originates from the primary and any secondary corpora lutea (CL) in response to the gonadotrophic actions of eCG. Oestrogen is a known stimulator of the vascular endothelial growth factor (VEGF) which promotes vasculogenesis and angiogenesis. This study assessed changes in luteal vascularity between days 14 to 50 of gestation. Fifteen pregnant mares in a clinical setting were examined at three time points (days 14–16, 20–30 and 40–50 after ovulation). At each examination pregnancy was confirmed and jugular venous blood samples assayed for progestagen and eCG concentrations. Ultrasonographic and Doppler examinations were performed using a SonoSite Micromaxx (SonoSite Ltd, Hitchin, UK) with a linear probe (5.0 10.0 MHz). Images were obtained in colour flow mode with the medium flow sensitivity pre-set. All images had a standard depth of 7.7 cm with a frequency of 3.92 MHz and pulse repetition frequency of 2.1 MHz. The colour image was frozen at peak systolic flow and recorded; acquisition was repeated 3 to 5 times for each CL. Images were analysed using ImageJ software (National Institutes of Health, Bathesda, USA).

The effects of embryo-uterine asynchrony on early embryonic development

Equine pregnancy has an extraordinary ability to tolerate embryo-uterine asynchrony. Indeed, while ruminant embryos will not survive negative asynchrony (i.e. the recipient ovulated after the donor) of more than 2 days [Lawson RAS,et.al, J Reprod Fertil, 1975;45:101-7], horse embryos will continue to develop in the uterus of a recipient that ovulated up to 7 days later [Wilsher S,et.al, Reproduction 2010;139:575-85.]. However, negative asynchrony of more than 4 days appears to compromise embryo development and survival and may therefore be a useful model to study embryo-maternal communication. This study examined the effects of negative uterine asynchrony on the development of equine embryos over a period of either 6 or 11 days. Embryos were recovered on day 8 after ovulation and transferred non-surgically to synchronous (day 8; donor-recipient difference 1⁄4 0 days) or asynchronous (day 3; donor-recipient difference 1⁄4 -5 days) recipient mares. The resulting conceptuses were recovered either 6 days later by uterine lavage or 11 days later using a video-endoscopically-guided net (n 1⁄4 5 per group in all cases). Prior to recovery, vesicle size was determined ultrasonographically; following recovery the embryonic disc and, for conceptuses recovered on day 11 post-ET, the embryo proper were identified, measured and staged in terms of primitive steak formation and indices of embryo development such as crown-rump length and somite

Two cases of equine pregnancy loss associated with Leptospira infection in England

INFECTION with Leptospira can cause abortions and stillbirths in domestic cattle, sheep and pigs, as well as in wild animals. It has also been well documented as a cause of...

The equine endometrial cup reaction: a fetomaternal signal of significance.

A remarkable feature of equine pregnancy is the development of the invasive trophoblast of the chorionic girdle and its formation of the gonadotrophin-secreting endometrial cup cells in early gestation. The details of this process have been revealed only slowly over the past century, since the first description of the endometrial cups in 1912. This centennial presents an opportunity to review the characteristics of the cells and molecules involved in this early, critical phase of placentation in the mare. The invasiveness of the chorionic girdle trophoblast appears to represent an atavistic attribute more commonly associated with the hemochorial placentae of primates and rodents but not with the more recently derived epitheliochorial placentae of the odd-toed ungulates. The nature of and raison d'être for the strong fetal signals transmitted to the mare by the endometrial cup reaction, and her responses to these messages, are the subject of the present review.

Expression of Inhibin/Activin Subunits in the Equine Uteri during the Early Pregnancy

The establishment of equine pregnancy is a unique and long process during which a series of physical and possibly biochemical interactions are required between the conceptus and uterus. In this study, we investigated the expression pattern of inhibin/activin subunits in the uterus during early pregnancy. The uteri from four adult mares on cyclic day 13 or pregnancy day 25 were obtained. Immunohistochemical experiments suggested that inhibin/activin subunits were immunolocalized in the luminal and glandular epithelium on pregnancy day 25. In addition, the inhibin α and inhibin/activin βB subunits were not detected, and inhibin/activin βA subunit was detected, in the luminal and glandular epithelium on cyclic day 13. Real-time polymerase chain reaction and Western blotting results for the inhibin/activin subunits suggested a significant increase in the expression of inhibin/activin subunit βB and a significant decrease in the expression of inhibin/activin subunit βA on pregnancy day 25 compared with those on cyclic day 13. Enzyme-linked immunosorbent assays suggested a significant decrease in the concentration of activin A in endometrium extracts from cyclic day 13 to pregnancy day 25. These results suggest that inhibins or activins synthesized in the uterus, as endocrine factors and necessary nutriments, have different expression patterns and may play different, important roles during early embryonic development of the equine.

Gene profiling of inflammatory genes in day 18 endometria from pregnant and non‐pregnant mares

Maternal recognition of pregnancy is a physiological process that primarily describes endometrial responses to a conceptus. Recognition of a conceptus prevents the release of prostaglandin F(2α) , thereby ensuring survival of the corpus luteum and continued progesterone production. Exactly how this occurs in the mare is poorly understood. Because prostaglandin F(2α) is a pro-inflammatory hormone, we hypothesized that differential gene expression in the endometrium at the time of maternal recognition reflects an anti-inflammatory event leading to decreased prostaglandin F(2α) secretion. Mares were inseminated, and endometrial biopsies were recovered from pregnant mares on Day 18 post-ovulation. In subsequent estrous cycles, mares were not inseminated and Day 18 post-ovulation endometrial biopsies were collected (non-pregnant control, matched per individual). Endometrial gene expression profiles were examined by screening an Affymetrix equine GeneChip containing probes specific for genes related to inflammatory processes. Microarray analysis revealed 118 genes that were up-regulated and 93 genes that were down-regulated (P < 0.001) at least 1.5-fold in the endometrium of pregnant versus non-pregnant mares. Quantitative, real-time RT-PCR confirmed the microarray results for three up-regulated genes homologous to TSC22D3, PPAPDC2, and KLF6, and three down-regulated genes homologous to ESR1, MARCKSL1, and EPSTI1 (P < 0.05). It is concluded that the presence of the equine embryo induces differential gene expression in the endometrium of Day 18 pregnant mares, and that these genes are associated with inflammatory processes and pathways involving cellular growth and proliferation. The results from this study provide important new insights into endometrial gene expression in response to early equine pregnancy.

Modulation of T‐cell Reactivity During Equine Pregnancy is Antigen Independent

Pregnant mares demonstrate a reduction in cytotoxic T lymphocyte (CTL) reactivity against cells from the breeding stallion. We investigated whether this effect is limited to activity against paternal major histocompatibility complex (MHC) antigens, and whether it occurs during MHC-compatible pregnancy. Mares were mated to carry MHC-compatible or MHC-incompatible pregnancies. CTL activity of these mares when pregnant and non-pregnant was measured against cells from horses with MHC haplotypes unrelated to the mare or breeding stallion. While carrying MHC-incompatible pregnancies, mares demonstrated reduced CTL activity against lymphocytes from third-party horses in addition to those from the breeding stallion. This effect was also observed in mares carrying MHC-compatible pregnancies. The decrease in maternal T-cell reactivity characteristic of normal equine pregnancy is not restricted to paternal alloantigen, and occurs during MHC-matched matings. This suggests that antigen-independent mechanisms may be responsible for this reduction in cell-mediated immune activity.

FoalinMare: insights inside the foaling mare

Jan Govaere, Katrien Martens and Aart de Kruif, together with their talented collaborators at the Department of Reproduction, Obstetrics and Herd Health at Ghent university (in cooperation with Utrecht university, Ohio State University and the University of Nantes), have produced an educational DVD that is intended mainly at students, vets and teachers; however, the DVD may also be suitable for biologists, horse’s breeders and owners. They used some of the best 3D animations I have ever seen. By means of this video support, they showed exactly what normally happens and what instead can deviate from physiologic during equine pregnancy and parturition; all in an effective and intuitive way......

Luteogenic and luteotropic effects of eCG during pregnancy in the mare

The role of eCG during pregnancy was evaluated through the study of the temporal relationships between changes in eCG and progesterone concentrations and the formation of supplementary corpora lutea (SCL) in mares impregnated with donkey semen (mule pregnancies) or with horse semen (equine pregnancies). Concentrations of eCG were higher (p<0.01) in equine than in mule pregnancies between weeks 6.5 and 13. Progesterone concentrations were higher in equine than in mule pregnancies between weeks 9 and 17. All animals developed at least one SCL, but more SCL accumulated during equine pregnancies than during mule pregnancies (1.9±0.2 vs 1.2±0.1; p<0.01). In equine pregnancies, the mares that formed a second SCL had higher eCG concentrations (p<0.05) during the two weeks preceding its formation than those mares remaining with only one SCL. Mares that formed a third SCL had higher (p<0.5) eCG levels than those remaining with one or two SCL. Mares with equine pregnancies that formed three SCL had higher progesterone concentrations (p<0.05) than those that formed only one or two SCL. No differences were found in progesterone or eCG concentrations between mares with mule pregnancies that accumulated different numbers of SCL during pregnancy (p>0.05). It is concluded that eCG stimulates both the development of new SCL and the function of existing CL. While these effects are clearly expressed in mares impregnated by horses, the low eCG concentrations during mule pregnancies reduce the impact of this hormone on CL formation and function.

Biosynthesis of oestrogen by the early equine embryo proper

The embryo proper in early equine pregnancy has recently been shown to have a remarkable capacity for metabolism of oestrogens. High concentrations of oestrogens in yolk-sac fluid could provide substrate for local metabolism in tissues of the embryo proper and this activity could have significance for early development. Due to the high level of oestrogen metabolism in the embryo proper we examined the possibility that it could also biosynthesise oestrogens. Conceptuses were collected in the fourth week of pregnancy (n=23) and the embryo was separated from extraembryonic tissues for incubation with [(3)H]androstenedione. Steroids were recovered from media by solid-phase extraction and eluted as unconjugated and conjugated fractions. Profiles of free and sulfoconjugated fractions, as well as the phenolic steroids extracted from them, were obtained by chromatography. Oestrone and oestradiol were seen clearly, indicating oestrogen biosynthesis, and the presence of more polar products, arising from metabolism of the primary oestrogens, gave further evidence that the embryo was capable of oestrogen biosynthesis. Aromatase activity was also demonstrated by detection of tritium loss, as (3)H(2)O, from incubations (n=3) with [1β-(3)H]androstenedione. It is suggested that its oestrogen biosynthesis may have significance for the remarkable development of the vasculature in the embryo proper at this stage.

The Relationship Between the Positive Identification of the Embryo Proper in Equine Pregnancies Aged 18-28 Days and its Future Viability: A Field Study

Early embryonic loss (EEL) negatively affects the reproductive efficiency of equine reproduction. A sign of future EEL is when the embryo proper (EP) fails to develop within the embryonic vesicle after 30 days of gestation. The earlier the identification of impending EEL, the earlier the mare can be rebred to allow a second chance of pregnancy. The objectives of this study were to determine the percentage of embryonic vesicles with a visible EP at 18-28 days of gestation and to study the association between the presence/absence of the EP at different days of gestation and the future viability of the pregnancy. A total of 1,256 pregnancies were identified and followed by transrectal B-mode ultrasonography 12-45 days post ovulation in mares of the same Thoroughbred farm. The identification of the EP was attempted once during days 18-28. Pregnancy reconfirmation was performed on days 35-45. The percentage of vesicles with an EP increased gradually from days 18 (2.8%) to 21 (86.9%) (P < .05). From day 20 onward, the EEL rate of mares with vesicles without an EP was significantly higher (P < .05) than that of vesicles with a positive identification of an EP. In conclusion, the EP of the equine vesicle can be identified reliably with B-mode ultrasonography in the majority of mares (>71%) on day 20 of gestation. The lack of a positive identification of an EP from day 20 onward is associated with a higher EEL rate.

Ultrasonographic features of the mule embryo, fetus and fetal-placental unit

The aim of this study was to establish baseline ultrasound data concerning the mule conceptus during gestation. Ten multiparous Trotter mares were artificially inseminated with chilled semen from an Amiatino jack donkey. Daily transrectal ultrasonography was carried out from the day of ovulation until Day 50 of gestation to determine the following: first detection of the embryonic vesicle (EV), mobility phase, EV diameter, day of EV fixation, changes in EV shape, date of yolk sac regression and embryo crown-rump length. Monthly ultrasonic assessments from Day 50 of gestation to term were carried out. These assessments included an evaluation of fetal well-being and the growth of the mule conceptus, which were monitored using the following variables: cardiac activity, fetal activity and presentation, fetal fluid echogenicity, combined thickness of the utero-placenta unit and fetal orbital and aortic diameter. Mule EV first detection was observed earlier (37% at Day 8) than that observed in the equine pregnancy. EV diameter at first detection was 4.6 ± 1.1 mm. At Day 10, 75% of EVs were detected. EV fixation occurred on Day 17.1 ± 1.1, with a mean EV diameter of 2.5 ± 0.2 cm. EV growth rate was 4.04 mm/day from Days 11 to 16, 0.4 mm/day from Days 16 to 28 and 1.78 mm/day from Days 28 to 45 of pregnancy. The embryo proper was first detected on Day 19.9 ± 1.9 (average length 2.4 ± 1.4 mm), and the embryonic heartbeat was first detected on Day 24 ± 2.4. The fetal carotid pulse was observed at six months of gestation and provided a good means by which to estimate fetal cardiac activity in advanced gestation. The fetal heart rate was recorded from Month 2 of gestation to term. The mean ± SD of the combined uteroplacental thickness was assessed at the cervical-placental junction and at the ventral abdomen in mares between Months 2 and 5 until term, respectively. An abnormal fetal-placental unit and fetal inactivity was observed in association with abortion. Mule-conceptus biometric measurements correlated significantly with the gestational age, and these data were used to predict an unusually large mule fetus, which might result in dystocia. In conclusion, we can assume that early diagnosis of pregnancy failure and assessment of fetal biophysical profile and growth charts could improve the chances of gestation completion in mule-pregnant mares. The early detection of mares at risk for an abnormal pregnancy or delivery may increase the success of prompt treatments, therefore preventing costly emergency procedures and allowing proper obstetrical and neonatal assistance.

The effects of an advanced uterine environment on embryonic survival in the mare

During embryo transfer (ET) the equine embryo can tolerate a wide degree of negative asynchrony but positive asynchrony of >2 days usually results in embryonic death. There is still confusion over whether this is due to the inability of the embryo to induce luteostasis or to an inappropriate uterine environment. To assess embryo survival and development in an advanced uterine environment. Embryo-uterine asynchrony, not the embryo's inability to induce luteostasis, is responsible for embryonic death in recipient mares with a >2 days chronologically advanced uterus. Experiment 1: Thirteen Day 7 embryos were transferred to the uteri of recipient mares with luteal prolongation, occasioned by manual crushing of their own conceptus, such that donor-recipient asynchrony was between +13 and +49 days. Experiment 2: Day 7 embryos were transferred to recipient mares carrying their own conceptus at Days 18 (n = 2), 15 (n = 2), 14 (n = 4), 12 (n = 4) or 11 (n = 4) of gestation. In addition, Day 8 embryos were transferred to 4 pregnant recipient mares on Day 11 of gestation. No pregnancies resulted following transfer of Day 7 embryos to recipients in prolonged dioestrus with asynchronies between +13 and +49 days. However, the use of early pregnant mares as recipients resulted in 5/20 (25%) twin pregnancies, 4 of which came from the transfer of a Day 8 embryo to a Day 11 recipient. All transferred embryos showed retarded growth, with death occurring in 4/5 (80%). The results emphasise the importance of an appropriate uterine environment for embryo growth and the inability of equine embryos to survive transfer to a uterus >2 days advanced even when luteostasis is achieved. It is possible that in normal, non-ET equine pregnancy, embryo-uterine asynchrony may account for some cases of embryonic death.

Production of Calcium Maintenance Factor Stanniocalcin-1 (STC1) by the Equine Endometrium During the Early Pregnant Period

A factor responsible for progression to pregnancy establishment in the mare has not been definitively characterized. To identify factors possibly involved in the establishment of equine pregnancy, the endometrium was collected from day 13 (day 0=day of ovulation) cyclic and day 13, 19 and 25 pregnant animals. From initial subtractive hybridization studies, a calcium regulating factor, Stanniocalcin-1 (STC1) mRNA, was found as a candidate molecule expressed uniquely in the pregnant endometrium. Endometrial expression of STC1 mRNA was noted on day 19 and was markedly increased in the day 25 gravid endometrium. STC1 protein was found in the extracts of day 25 gravid endometrium and immunochemically localized in the uterine glands. In addition, STC1 protein was detected in uterine flushing media collected from day 25 pregnant mares. High concentrations of estradiol-17 β (E(2)) were detected in day 25 conceptuses. E(2) levels were much higher in the gravid endometrium than in other regions, whereas progesterone levels did not differ among the samples from different endometrial regions. Expression of STC1 mRNA, however, was not significantly upregulated in cultured endometrial explants treated with various concentrations of E(2) (0.01-100 ng/ml) with or without 10 ng/ml progesterone. These results indicate that an increase in STC1 expression appears to coincide with capsule disappearance in the conceptus, and suggest that STC1 from the uterine glands likely plays a role in conceptus development during the pregnancy establishment period in the mare.

XX/XY Blood Lymphocyte Chimerism in Heterosexual Dizygotic Twins from an American Bashkir Curly Horse. Case Report

Cytogenetic analysis and microsatellite genotyping were conducted on a pair of phenotypically normal dizygotic heterosexual equine twins of the American Bashkir Curly breed. The animals had a mixture of 64,XX and 64,XY cells in blood lymphocytes, with their own cells being predominant. Therefore, the 64,XX cells comprised 81% of the lymphocyte population in the female twin and 64,XY comprised 79% in the male twin. Blood chimerism was confirmed by genotyping 30 microsatellite markers. Of these, 15 microsatellites showed the presence of three alleles and all four parental alleles in the blood lymphocytes for both animals. No chimerism was detected in the genomic deoxyribonucleic acid isolated from hair follicles. These results are in agreement with earlier observations that vascular anastomoses can infrequently occur during equine multiple pregnancies resulting in blood lymphocyte chimerism without significant effect on the phenotype.

Maternal Immune Responses to Trophoblast: The Contribution of the Horse to Pregnancy Immunology

The horse has proven to be a distinctively informative species in the study of pregnancy immunology for several reasons. First, unique aspects of the anatomy and physiology of the equine conceptus facilitate approaches that are not possible in other model organisms, such as non-surgical recovery of early stage embryos and conceptuses and isolation of pure trophoblast cell populations. Second, pregnant mares make strong cytotoxic antibody responses to paternal major histocompatibility complex class I antigens expressed by the chorionic girdle cells, permitting detailed evaluation of the antigenicity of these invasive trophoblasts and how they affect the maternal immune system. Third, there is abundant evidence for local maternal cellular immune responses to the invading trophoblasts in the pregnant mare. The survival of the equine fetus in the face of strong maternal immune responses highlights the complex immunoregulatory mechanisms that result in materno-fetal tolerance. Finally, the parallels between human and horse trophoblast cell types, their gene expression, and function make the study of equine pregnancy highly relevant to human health. Here, we review the most pertinent aspects of equine reproductive immunology and how studies of the pregnant mare have contributed to our understanding of maternal acceptance of the allogeneic fetus.

Evaluation of genes involved in prostaglandin action in equine endometrium during estrous cycle and early pregnancy

The aim was to evaluate expression of genes involved in the biosynthesis of prostaglandins (PTG), Prostaglandin H Synthase-1 (PTGS1) and PTGS2, PGF synthase (PTGFS), and PGE synthase (PTGES), PGF receptor (PTGFR), PGE receptors (PTGER2 and PTGER4), prostaglandin transporter (SLCO2A1) and hydroxyprostaglandin dehydrogenase-15 (HPGD). Endometrial biopsies were obtained from mares on day of ovulation (d0, n = 4), late diestrus (LD, n = 4), early luteolysis (EL, n = 4) and after luteolysis (AL, n = 4) during the cycle. Stages of the cycle were confirmed by plasma progesterone concentrations measured daily and ultrasound examinations. Biopsies were also taken on days 14 (P14; n = 4), 15 (P15, n = 4), 18 (P18, n = 4) and 22 (P22; n = 4) of pregnancy. Relative mRNA expressions were quantified using real-time RT-PCR. A mixed model was fitted on the normalized data and least significant difference test ( α = 0.05) was employed. Expression of PTGS1 mRNA was low throughout the estrous cycle and early days of pregnancy, but upregulated on P18 and P22. PTGS2 expression was increased on EL, but it was suppressed by pregnancy on P15, P18, and P22. PTGFS expression was upregulated in both cyclic and pregnant mares compared to d0 and its level was the highest on LD. PTGFR expression was transiently increased on LD and EL and was suppressed during early pregnancy. Both PTGES and PTGER2 expressions were increased on LD, EL, and early pregnancy, but were decreased after the luteolysis in cyclic mares as they remained high on P18 and P22. PTGER4 expression did not change throughout the cycle and early pregnancy. Levels of HPGD and SLCO2A1 were significantly increased only on P22. In conclusion, PTGS2 expression increases around the time of luteolysis and concurrent upregulation of PTGFS and PTGES indicates that equine endometrium has increased capability of PTG production around the time of luteolysis. However, pregnancy reduces PTGS2 expression, but maintains the high levels of PTGES during early pregnancy along with PTGER2 while PTGFR expression was suppressed. These findings suggest that possible luteotrophic action of PGE 2 is required in early equine pregnancy. PTGS1 is only upregulated later in the early pregnancy suggesting that it is not involved in luteolysis, but could be the main PTGS enzyme at this time during early pregnancy. An increase in HPGD and SLCO2A1 levels on P22 indicates a tight regulation of PTG action by pregnancy.

Evidence for NK cells in equine endometrial cups (43.29)

Abstract Placentation during equine pregnancy is distinguished by a unique subset of trophoblast cells that invade the endometrium and form discrete structures termed the endometrial cups. Shortly after the trophoblasts invade, there is a dramatic infiltration of maternal leukocytes which accumulate around the cups. Our lab has previously characterized the infiltrating leukocytes as primarily CD3+ cells with substantial CD4+ and CD8+ subpopulations. Here, we identify expression of the NK cell marker NKp46 in peripheral blood mononuclear cells (PBMC) and endometrial cup lymphocytes (ECL). PCR primers were developed to detect the equine homolog of NKp46 and expression was detected in resting and stimulated lymphocytes using standard PCR. Quantitative real-time PCR primers and standards were used to quantitate expression in CD3+ and CD3- sorted lymphocytes. NKp46 gene expression in CD3- lymphocytes was 10-fold higher than in CD3+ cells. Levels comparable to CD3-depleted lymphocytes were detected in frozen tissue sections of endometrial cups. The highest levels of expression were detected in leukocytes isolated from the endometrium surrounding the cups of five mares during early pregnancy (days 43-46). NKp46 expression levels were 7-43 fold higher in ECL compared to paired PBMC (p=0.04). Coincident CD3γ expression was lower in all ECL compared to paired PBMC (p=0.03). These data provide the first evidence for the presence of NK cells in the equine endometrium during pregnancy.