Amorphus globosus foetuses in Polish Holstein cattle: anatomical, histological, and genetic studies

Amorphus globosus (ag) foetuses are rarely reported in domestic animals, but the majority of recently documented cases were found in cattle, as well as in goats, sheep, and buffaloes (1, 5, 7, 8, 9, 10, 12, 13, 14, 16, 18, 19, 20, 22, 23, 24, 25). The customary nameamorphus globosus describes the shape of the foetus and the absence of clear differentiation of phenotypic body parts. The lack of an independent circulatory system expressed in the Latin nameacardius amorphus is a characteristic feature of the defect. The gross appearance of the teras is characterised by a round or spherical shape, generally without differentiated body parts, and is covered with hairy skin to a varying degree. The inner mass consists of tissue, some mesodermal (connective, muscle, adipose and adenoid, and cartilaginous and/or bone) and some ectodermal (1, 5, 13, 14, 22). They do not form any anatomical structures (or functional organs), but an exception to this has been noted, which included the presence of primordial germs of extremities (1).

Cases of amorphous foetuses in animals suggest that they usually do not occur by themselves but nearly always coexist with a living or dead co-twin or with co-multiples, which do not show any phenotypic malformations. Terata were usually found in cases of dystocia which were a result of incorrect foetal position. Normal foetuses and their coexistingag foetus may be of the same or opposite sexes (1, 10, 24, 25). However, it has been suggested that the female sex is predominant among the properly developed foetuses (14, 17, 18, 19, 22). There are also reports of the presence of XX/XY chimerisms and other chromosomal abnormalities inag co-twins which originated from the transfer of two embryos (16, 19).

Since the origin ofag foetuses is obscure, we undertook a comprehensive description, including morphological, histological, and genetic analysis of four novelag cases which originated from multiple pregnancies of three Polish Holstein cows.

Incidences. Four amorphous foetuses were delivered by three cows:

Cow A: a three-year-old 80% Holstein Friesian (HF) after the second calving. This cow gave birth to a live bull-calf (Am) weighing 33 kg and anag teras (A1) with a spherical shape of 12.0 × 12.0 cm, weighing 533 g and with well-developed skin thinly covering the entire surface. Unpigmented skin occupied approximately 30% of the total area and was covered with long white thin hair (approximately 1.5 cm in length) in contrast to the pigmented skin area. A strip of 5cm maximal width which narrowed at the ends closer to the umbilical cord divided the pigmented skin surface area into two unsymmetrical parts. The preserved thin umbilical cord section had a thickness of approximately 1 cm and a length of approximately 25 cm. The calf was delivered after 272 days of gestation and the expulsion of the placenta occurred spontaneously.

Cow B: a five-year-old HF after the third calving. Parturition produced two apparently normal dead bulls, without any signs of developmental disorders, and an elongated oval shapedag (B1) measuring 15.3 × 8.5 cm, weighing 390 g, and covered with thin skin. Most of its surface was pigment-free (5% of the skin had black pigment) with scant thin white hair strands. The calves were delivered after 272 days of gestation but required obstetric intervention due to obstruction caused by impaction of both foetuses. The postnatal examination revealed the presence of a spherical malformed co-multiple which was removed from the uterine cavity along with a part of the placenta. The retained placenta was noted after delivery.

Cow C: a four-year-old HF after the third calving. The animal delivered two live calves (a heifer (C-f) of 32 kg and a bull (C-m) of 33 kg) along with twoag of dissimilar sizes and shapes. The larger (C1) was spherical, measuring 15.7 × 15.0 cm, weighing 769 g and with well-developed skin covered with thick, black hair (approximately 1.5 cm long) covering approximately 95% of its surface. An area of unpigmented skin of approximately 5 cm² was found peripherally from the umbilical cord and a smaller area located right around the umbilical cord was covered with sparser and slightly longer white hair. On the surface, there were no natural orifices or primordial body parts that would suggest its anterior and posterior (cranial and caudal) ends. The preserved portion of the umbilical cord had a thickness of approximately 2.5 cm and a length of approximately 30 cm. There were no cotyledons detected. The second amorphous foetus (C2) was made up of two spherical structures with dimensions of 8.7 × 8.5 cm and 5.1 × 3.8 cm and a combined weight of 344 g; they were joined by a narrower section of tissue with a width of 3.8 cm. Both were covered with well-developed skin densely covered with hair and pigmented over most of their surface. The intact portion of the umbilical cord had a diameter of approximately 5.5 cm and a length of 15 cm; in the part still in contact with the foetal mass, there was a fatty body measuring 5.8 × 4.4 cm and weighing 84.5 g. Two cotyledons were located more peripherally. Delivery was aided with the use of a calf puller after 273 days of gestation. The examination of the uterine cavity confirmed the existence of additional amorphous structures, which were removed mechanically. A retained placenta was noted in the course of postnatal care.

Histology. Tissue samples were collected during autopsy. They represented a cross-section of three layers, including the skin and the tissues located deeper, as far as to the central zone of the amorphous foetus. Samples were fixed in 10% buffered neutral formalin. The tissue was embedded in paraffin, sectioned at 5 μm, and stained with haematoxylin and eosin (HE).

Cytogenetic and molecular genetic studies. Chromosome preparations were obtained from a short-term lymphocyte culture. Briefly, heparinised blood was cultured in RPMI 1640 medium, supplemented with foetal calf serum (15%), an antibiotic-antimycotic mixture (1%), and phytohaemagglutinin for 72 h at 37°C. After colcemid treatment, a standard harvesting procedure including hypotonic and fixative steps was applied. Chromosome slides were conventionally stained with Giemsa solution and analysed under an E600 Eclipse microscope (Nikon, Japan). Bovine sex chromosomes were identified based on their bi-armed morphology, i.e. a large submetacentric X chromosome and a small metacentric Y chromosome. For each individual 50 metaphase spreads were evaluated.

Genomic DNA was isolated from tissue samples of the abnormal foetuses (A1, B1, C1, and C2) using a Genomic Mini kit (A&A Biotechnology, Poland) and from blood leukocytes of their healthy siblings (cases A and C) using a Blood Mini kit (A&A Biotechnology). PCR reactions were used to reveal the presence of Y-linked genes SRY andAMELY) and an X-linked gene AMELX). To detect theSRY gene, a PCR reaction was performed with primers (5′-AAGAACAACTTAT GAATAGCACCA-3′ and 5′-TTAAGTCGCAGGTGA AACTGT-3′) resulting in an 851 bp amplicon. To detect theAMELX andAMELY genes the PCR reaction was carried out with primers (5′-CAGCCAAACCTCC CTCTGC-3′ and 5′-CCCGCTTGGTCTTGTCTGT TGC-3′) amplifying a 280 bp fragment of theAMELX gene and a 217 bp fragment of theAMELY gene. The PCR conditions were initial denaturation at 94°C for 5 min, followed by 35 cycles of denaturation at 94°C for 30 s, primer annealing at 60°C or 64°C for 30 s (for theSRY andAMELX/AMELY genes, respectively), primer elongation at 72°C for 1 min, and the final elongation at 72°C for 5 min. The resulting PCR fragments were visualised using agarose gel electrophoresis. DNA profiles were established for the abnormal foetuses (A1, C1, and C2) and their healthy siblings (Am, Cm, and Cf) using 17 microsatellite markers (TGLA227, BM2113, ETH10, SPS115, SPS113, RM067, TGLA126, TGLA122, INRA23, BM1818, ETH3, ETH225, BM1824, CSRM60, MGTG4B, CSSM66, and ILSTS006) by Dr Van Haeringen Laboratory (VHL Genetics, the Netherlands).

Allag foetuses were classified in the groupholoacardius amorphus (anideus). Macroscopic comparisons focused attention on four main differences between the studiedag: size/weight, shape, hair density, and differences in the appearance of the umbilical cord stump. The biggestag were observed in the cases of the double and quadruple pregnancies, in which the amorphous forms came from homozygous pregnancies, as confirmed by genetic testing. The case of the birth of twoag was identified as a homozygous triple pregnancy (Table 1), whereby both amorphous foetuses differed substantially in shape, size, and weight. Although similar in size and shape, the large, spherical amorphous forms had different hair densities (Figs 1 and 3). The lowest hair density was observed in B1 (Fig 2), in which hair was only residual, and the highest in C1 and C2 (Figs 3 and 4). In the case of C2, the histological examination revealed a further higher density of hair in the pigmented skin area. Lower hair density was associated with its more balanced and advanced development (fewer hair follicles at various stages of development, and well-developed structure of the hair and follicles).

Fig. 1

Fig. 2

Fig. 3

Fig. 4

The appearance of the intact fragments of the umbilical cord remnants varied (Figs 1–4). The thinnest stump was found in A1, while the longest were in B1 and C1. The umbilical cord stump of C2 was short, but in the immediate vicinity of the amorphous mass outside the fatty body, it contained two cotyledons.

Dominant in A1 were a large variety of early forms of hair development (Fig. 5). Asynchronicity of their development and the presence of the same forms of development at different distances from the epidermis was observed. B1 was an isolated case, where very thin unpigmented hair dominated. Circular cross-sections of numerous channels and hair sheaths in the layers abundant in keratin,i.e. the epidermis and dermis, indicate more horizontal rather than perpendicular hair development.

Fig. 5

On the border between the layers of keratin, separated from the deeper tissue, there were either no cells observed or they occurred singly (an epidermal cell layer was lacking). Moreover, the tissues lying deeper were hypocellular, with the exception of regions in the immediate vicinity of the very early forms of hair development. A similar structure was characterised by a wall cavity located in the amorphous mass (Fig. 6), in which elements of the structure of the hair occurred less frequently. In the separating layer of keratin and deeper tissues, cells with the characteristics of kernel defragmentation were found.

Fig. 6

The histopathological examination of selected areas of the parenchyma of the amorphous foetuses revealed tissues of meso- and ectodermal origin. In all cases, fatty, mesenchymal tissue with varying density of cells and matrix as well as muscle dominated (Figs 7 and 8). Large fluid passages and vessels of various wall organisations and clusters of lymphoid tissue occurred in the mass of the parenchyma (Fig. 8). Figures 9 and 10 illustrate irregular spaces with features of epithelial lining as well as the chondral elements similar in structure to long bones observed in C2. In the parenchyma of B1 there was an oval sponge-like bone mass with approximate dimensions of 3.0 × 2.5 × 1.5 cm (no image) and in adjacent parenchyma areas corresponding to skeletal muscle.

Fig. 7

Fig. 8

Fig. 9

Fig. 10

The chromosome complement was analysed in the normal siblings of amorphic foetuses (A-m, C-m, and C-f) in order to determine their sex chromosomes and search for leukocyte chimerism (XX/XY), which is very common in heterosexual twins. It was found that in all viable calves, only one cell line was identified and the sex chromosome complement was as follows:

A-m (60, XY), C-m (60, XY), and C-f (60, XX) (Figs 11 and 12, Table 1).

Fig. 11

Fig. 12

A comparative study of microsatellite polymorphism of the amorphous foetuses (A1, C1, and C2) and their normal siblings (A-m, C-m, and C-f) showed that they derived from the same zygotes. Amorphous foetus A1 and its normal sibling had the same genotype at 17 microsatellite loci and Y-linked SRY andAMELY) and X-linked AMELX) genes (Figs 13 and 15). The C1 and C2 amorphous foetuses had the same genotype at the studied microsatellite loci as their normal female sibling (C-f). Moreover, in both foetuses only theAMELX gene was detected (Figs 14 and 16). In the foetus B1 two Y-linked SRY andAMELY) and an X-linked AMELX) gene were found. Unfortunately, molecular analysis of the two stillborn male full siblings was not possible. The results for cow A’s product of conception and offspring showed that amorphous foetus A1 and normal male calf (A-m) originated from the same zygote. Similarly, the C1 and C2 amorphous foetuses and the normal female calf (C-f) also originated from the same zygote.

Fig. 13

Fig. 14

Fig. 15

Fig. 16

Most cases ofag described in the literature as well as cases from personal experience were discovered at the point of obstetric assistance or during medical intervention after a retained placenta had come to notice. All the deliveries took place after the normal duration of gestation. An amorphous foetus has also been documented in a cow that died in the eighth month of a hexafoetal pregnancy (10). Due to its low weight the parasitical foetus is rarely the cause of lower birth weight of the co-multiples. This can be confirmed by the similar body weight of the two live newborns, one of which was a homozygous co-multiple of the two amorphous foetuses (case C). In the rest of the cases of pregnancy withag described in this paper, the well-developed embryos had a mass within the range of normal values.

Our observations did not confirm the opinion that the female sex is more common among co-twins ofag (22). From our case study, it was the male sex that predominated among the animals without phenotypic defects, while the sex ofag varied determined on the basis of genetic tests.

Records of congenital disorders indicate that occurrences ofag in animals are quite rare (4). In humans, their incidence is estimated at 1 per 35,000 pregnancies or 1% of monozygotic twin pregnancies, pointing to the possibility of coexistence in multiple pregnancies (2, 3, 15). Amorphous foetuses have living tissue at the point of birth, hence in the absence of the heart, the supply of oxygen and nutrients requires communication with the vascular system of a co-twin. Blood flow inag in homozygous pregnancies is possible thanks to collateral circulation and/or arterio-arterial anastomoses of the chorion, whereas in heterozygous pregnancies this theoretically only takes place through anastomoses. Thus, the degree of blood supply can be the result of various pathological developmental variants of the vascular system.

The causes of acardia include insufficient embryonic mesoderm, anastomoses between the vessels of the umbilical cord, and disturbances in the return flow of blood from the placenta to the foetus (5). In the present day, a twin reversed arterial perfusion is indicated as the main cause of the defect (11). It is a rare complication of monochorionic pregnancy that involves countercurrent flow of deoxygenated blood from the umbilical cord arteries of the foetus through its branch connected to the umbilical artery of the twin with acardia and its return along the umbilical cord vein back to the placenta. Such advanced defects in the construction of vessels in the ruminant placenta have not been confirmed.

Developmental disorders arising as a result of TRAP are called disruption or breakage and they involve lesions in the preexisting tissue structures, which had been developing properly up to a certain point. Amorphous foetuses of ruminants show an extremely low degree of tissue organisation (1, 13, 14). However, during histopathological tests, certain features were found to confirm the theory of disruption. In all cases, the keratin layer tended to be separate from the deeper underlying tissue. Its presence in the skin was observed in all cases, but in some of them it was expanded and occurred despite the absence of or very sparse presence of skin cells. In B1, similarly to skin, the cavity lining contained developed hair, however, there were no cellular elements in the hair sheaths. Also the buds of roots and hair canals were hypocellular. Beneath the keratin layer, decay of cell nuclei was observed. There were large areas of the matrix of connective tissue with few cells found in the parenchyma. These features were most clearly marked in the case of B1, deriving from a triple pregnancy which resulted in the delivery of two dead coexisting foetuses. In C2 free spaces indicated only partial epithelialisation of the lining.

In the literature there is a lack of conclusive evidence on the possibility of the development of an amorphous foetus in a multiple pregnancy of mixed sexes. However, it does describe two cases of twin pregnancies withag, which were preceded by double ovulation confirmed by the presence of two corpora lutea on the ovaries at the time of birth, and it also reports the occurrence of chimerism in female co-twins as well as a case of an amorphous foetus delivered after the transfer of two embryos afterin vitro fertilisation (16, 19, 22, 25).

Our molecular analysis clearly showed that the studied amorphous foetuses had genotypes at 17 microsatellite loci identical to those of the viable co-twins, thus, their origin was monozygotic. Interestingly, a dizygotic origin ofag was recently reported by Weberet al. (25), who genotyped the samples.

The degree of tissue organisation ofag was partly linked to the appearance of the surviving fragments of the umbilical cord. A very thin or long umbilical cord stump was observed in theag with the lowest degree of tissue differentiation (A1/C1), while in the case of the umbilical cord with cotyledons, even though the amorphous foetus was the smallest, it revealed the highest diversity of tissues (C2). This indicates that the size of theag does not reflect the progress of the development of tissue. Only B1 went counter to the umbilical cord and tissue differentiation relationship, because despite a long thick umbilical cord, bone tissue indicated a more advanced development of mesodermal derivatives in the foetal mass. In this case, the presence of the cavity in the amorphous mass and the similarity of the histological structure of its wall with the skin indicate the disordered migration of embryonic primordia of the ectoderm. The nuclear disintegration in cells in the lining of the cavity and the lack of cellular elements in the immediate vicinity of exfoliating keratin layers on the surface of B1 (no skin) indicate degenerative regressive processes.

Sometimes co-twins of amorphous foetuses are born dead (1, 25). They usually die as a result of becoming stuck in the reproductive tract, prolonging the delivery and hypoxia. The available literature lacks data on the impact ofag in animals on growth and survival of co-multiples. The heart of the foetus with no defect bears the functional load resulting from the need to supply blood to both itself and theag. Hypoxia of tissues may be the reason for increased neovascularisation of amorphic foetuses (6). A decrease in vascular resistance resulting from the need to maintain blood pressure and oxygenation in the parasitic foetus systematically increases the workload on the heart of the “pumping” foetus. In human monochorionic foetuses from twin pregnancies withag we can observe congestive heart failure. It may result in intrauterine growth retardation, foetal death, stillbirths, and increased mortality during the post-parturient period. The impossibility of performing an autopsy on the stillborn foetuses of cow B and only one description of the changes that occur following the autopsy of a stillborn heifer in the literature make it impossible to entertain the coexistence described in cattle (25).

Our investigation proved that the studied foetuses originated from monozygotic twins in which the co-twins were normally developed. The histological analysis showed a low level of development of tissues of meso- and ectodermal origin, as well as features of degrading patterns.