Patterns of conceptus development and of progesterone concentrations in maternal blood preceding spontaneous early pregnancy failure in mares.

Sixteen cases of spontaneous pregnancy loss (11 of singletons and five of pairs of twins) are described. The losses occurred between gestation Days 13 and 25 in 12 mares being monitored almost daily by transrectal ultrasonography (for measurement of conceptus growth) and blood sampling (for determination of maternal plasma progesterone concentrations as evidence of luteolysis) in experimental studies of early pregnancy. In 10 of the 16 cases the uterus was flushed and eight conceptuses were recovered for morphological assessment. Five of the 11 losses of singletons occurred before Day 16 and, with one exception, were preceded or accompanied by luteolysis. The remaining six singleton pregnancies failed after Day 16, with two cases evidencing luteolysis beforehand. Thus, overall, 6/11 singleton losses were associated with luteolysis while 5/11 were not. The five cases of simultaneous loss or degeneration of twin conceptuses all occurred on Day 19 or 20, preceded by luteolysis in only one case. These observations suggest that while the causes of spontaneous early pregnancy failure are multifactorial, luteolysis might contribute to the problem more often than has been previously contended.

Morphology of twin and triplet equine conceptuses during weeks 3 and 4 of pregnancy.

Twin ovulations are common in horses, but twin pregnancies are rarely carried to term. Theories of how one or both twins is/are naturally eliminated in early pregnancy, termed 'embryo reduction', have been based on ultrasonographic, not morphological, studies. Here we describe conceptuses recovered transcervically between Days 15 and 28 from 31 twin and two triplet pregnancies. Signs of contact between conceptuses were deduced from those seen in one pair that remained attached by their capsules on Day 18. Signs were found on capsules in two of 10 pairs before or during fixation (immobilisation) at Days 16-17 even though contact had not been seen by ultrasound. After fixation, the signs became stronger in seven of nine unilateral pregnancies, indicated adhesion between pairs and included effects on the vitelline circulation and/or degeneration of one twin. Conceptuses recovered from five of seven unilateral twin pregnancies after the time of capsule disruption (~Day 21) evidenced embryo reduction; in the two surviving pairs, attachment between twins was near the trilaminar/bilaminar yolk-sac wall border. The findings are consistent with the notions that: (1) the capsule plays a role in initiating adhesion between twins; and (2) twin survival depends on an unencumbered trilaminar yolk-sac wall and a functional vitelline circulation.

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 [1ß-(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.

Relationship between the timing of prostaglandin-induced luteolysis and effects on the conceptus during early pregnancy in mares.

To advance the understanding of early pregnancy and pregnancy failure in horses, this study determined how luteolysis induced by cloprostenol (an analogue of prostaglandin F2α) affects conceptus development. Mares were injected on Days 12, 14, 16 or 18 of pregnancy with either cloprostenol (treatment groups, total n=83 pregnancies) or saline (controls, n=81), and growth of the conceptuses was monitored and compared by daily ultrasonography until they were collected transcervically on Days 15-22, 1-4 days after the injections. The comparisons were extended in the recovered conceptuses by counting somites, measuring the volume and osmolality of yolk-sac fluid and its concentrations of proteins, estrone sulfate and progesterone, and by assessing the morphology of the capsule and vascular system. When luteolysis was initiated on or before Day 16, most pregnancies survived until the time of collection and the conceptuses in respective treated and control groups on Days 15-20 were very similar except for some effects of treatment on the capsule and vascular development. In contrast, after luteolysis was initiated on Day 18, abortion often ensued within 3 days and most conceptuses collected had degenerated, therein constituting a predictable system in which to study the pathogenesis of a particular cause of pregnancy failure.

Estrogen metabolism by the equine embryo proper during the fourth week of pregnancy.

Estrogen production by the trophoblast is considered important in early equine pregnancy and leads to high concentrations in yolk-sac (Y-S) fluid. The embryo proper is a potential site for their action. We examined estrogen metabolism in the embryo proper because some actions of estrogens are derived from locally formed metabolites. The embryo proper, as well as separated extraembryonic tissues, of conceptuses collected about day 25 of pregnancy, were incubated with (3)[H]-estrone (E(1)) and (3)[H]-estradiol (E(2)). Steroids were recovered from media by solid-phase extraction and eluted separately as unconjugated and conjugated fractions. Profiles of free and sulfo-conjugated fractions were obtained by HPLC. Some differences and similarities were noted for the embryo proper as compared to the extraembryonic tissues. No reduction of E(1) to E(2) was noted for the embryo proper and allantois, but some was seen with the bilaminar Y-S wall. Less conversion of E(2) to E(1) occurred in the embryo proper than in the extraembryonic tissues. Profiles for hydrolyzed sulfates from incubation of the embryo proper were very similar for both substrates, mainly with E(1) present. Thus, low levels of reductase and high levels of oxido- activities were apparent for the 17beta-hydroxysteroid dehydrogenase enzymes. Further evidence of an active role for the embryo proper was seen as minor, polar products, and an unknown compound eluting between E(2) and E(1). These findings show, for the first time, that the embryo proper can metabolize estrogens that are found in Y-S fluid - a function of potential significance at this stage in its development.

Electrolyte distribution and yolk sac morphology in frozen hydrated equine conceptuses during the second week of pregnancy.

To investigate how equine conceptuses expand rapidly despite the hypo-osmolality of their yolk sac fluid, 18 conceptuses, aged 8-12 days and 0.8-10.0 mm in diameter, were examined by cryoscanning electron microscopy and energy dispersive X-ray microanalysis to determine the distribution of Na, Cl and K in their fluids. No osmotic gradient was found between central and peripheral yolk sac fluid. In conceptuses > or = 6 mm in diameter, the concentrations of both Na and K in the subtrophectodermal compartments were higher than those determined previously in uterine fluid, supporting the concept of osmotic intake of fluid from the uterine environment as far as the compartments. However, electrolyte concentrations in the compartments consistently exceeded those found in the yolk sac, making it likely that 'uphill' water transport, rather than a purely osmotic uptake, is involved in yolk sac fluid accumulation. We also speculate that capsule formation could actively contribute to conceptus expansion and thereby to fluid intake.

Desialylation of core type 1 O-glycan in the equine embryonic capsule coincides with immobilization of the conceptus in the uterus.

During the second and third weeks of pregnancy, the equine conceptus expands rapidly while it is enclosed within a glycan capsule. Around day 16 of gestation, the conceptus loses its mobility in the uterus by a process termed 'fixation', coinciding with various changes in the capsule. Here, we compared the structure of the carbohydrate moieties expressed by the capsule during pre- and post-fixation periods. The glycan structures were studied by chemical analyses in combination with mass spectrometry. Capsule material from conceptuses collected before fixation (days 13-16) was observed to carry a sialylated core type 1 O-linked glycan, Neu5Ac-(2-->3)-Gal-(1-->3)-GalNAc-(1-->Ser/Thr. By comparison, analysis of post-fixation capsules (days 17-19) revealed a desialylated core type 1, Gal-(1-->3)-GalNAc-(1-->Ser/Thr. The equine embryonic capsule also furnished 4-substituted GlcNAc, 4-substituted Glc and 2,3,4,6-tetrasubstituted Glc residues, the concentrations of which did not change between pre- and post-fixation stages. The loss of sialic acid from the sialylated core type 1 in the capsule appears to be directly related to successful fixation of the conceptus, and thus critical to the continuance of pregnancy in horses.

Estrogen metabolism in the equine conceptus and endometrium during early pregnancy in relation to estrogen concentrations in yolk-sac fluid.

Because estradiol (E(2)) production by the early equine conceptus is considered crucial to the establishment of pregnancy, the amounts of E(2), estrone (E(1)), and their sulfates (E(2)S, E(1)S) were measured by RIA in yolk-sac fluid of 63 conceptuses collected by transcervical lavage over the period of 11-26 days after ovulation. Amounts increased significantly with age of conceptus, especially for E(1)S. Then, the metabolism of E(2), which may be highly relevant for its action, was examined in the conceptus and endometrium over the period when the conceptus ceases to migrate within the uterus. Eleven conceptuses collected mainly on Days 12, 15, and 18, with endometrial biopsy samples taken immediately thereafter, were used for steroid metabolic studies. Trophoblastic and endometrial tissues were incubated with [(3)H]-labeled E(2) or E(1), and with [(14)C]-E(1) in one experiment. Steroids were recovered from the media by solid-phase extraction (SPE) and eluted separately as unconjugated and conjugated fractions. Conjugation increased from Day 12 for the trophoblast (more so by bilaminar than trilaminar tissues on Day 18) and was much greater for endometrium, with almost all as sulfoconjugates. HPLC profiles of free and sulfate fractions were obtained from a gradient of acetonitrile/water. Interconversion (E(2) right harpoon over left harpoon E(1)) by trophoblast varied with development; it favored E(2) in older conceptuses, more in bilaminar than trilaminar tissues. Some more polar products were also noted, with loss of tritium seen as [(3)H](2)O at SPE, and confirmed by HPLC in a second system with authentic reference steroids. Almost all radioactivity in the endometrium was present as E(2) in both free and sulfate fractions. It was concluded that local metabolism of E(2) is quantitatively significant and may play an important role in the actions of the large amounts of estradiol produced by the early equine conceptus.