Application of an artificial intelligence for quantitative analysis of endothelial capillary beds in vitro.

Objective: BACKGROUND: The use of endothelial cell cultures has become fundamental to study angiogenesis. Recent advances in artificial intelligences (AI) offer opportunities to develop automated assessment methods in medical research, analyzing larger datasets. Objective: OBJECTIVE: The aim of this study was to compare the application of AI with a manual method to morphometrically quantify in vitro angiogenesis. Objective: METHODS: Co-cultures of human microvascular endothelial cells and fibroblasts were incubated mimicking endothelial capillary-beds. An AI-software was trained for segmentation of endothelial capillaries on anti-CD31-labeled light microscope crops. Number of capillaries and branches and average capillary diameter were measured by the AI and manually on 115 crops. Objective: RESULTS: The crops were analyzed faster by the AI than manually (3 minutes vs 1 hour per crop). Using the AI, systematically more capillaries (mean 48/mm2 vs 27/mm2) and branches (mean 23/mm2 vs 11/mm2) were counted than manually. Both methods had a strong linear relationship in counting capillaries and branches (r-capillaries = 0.88, r-branches = 0.89). No correlation was found for measurements of the diameter (r-diameter = 0.15). Objective: CONCLUSIONS: The present AI reduces the time required for quantitative analysis of angiogenesis on large datasets, and correlates well with manual analysis.

Microvascular changes following exposure to iodinated contrast media in vitro. A qualitative comparison to serum creatinine concentrations in post-cardiac catheterization patients.

INTRODUCTION: Contrast-associated acute kidney injury (CA-AKI) is characterized as a loss of renal function following radiological contrast media administration. While all contrast media induce variable changes in microvascular endothelial cells in vitro, only few studies report clinical significance of their findings. A comprehensive assessment of the effect of iodinated contrast media on the renal function in vitro and in vivo is essential. The aim of our study was to morphometrically quantify the effect of two different contrast media (Iobitridol and Iodixanol) on vascular endothelial capillaries in vitro and to analyze their effect on the renal function of patients who underwent cardiac catheterization including the intra-arterial administration of contrast media, by measuring serum creatinine concentration (SCr), a byproduct of muscle metabolism, primarily excreted by the kidneys. Our hypothesis suggests that conducting a qualitative comparison of both outcomes will enable identification of differences and similarities between in vitro and in vivo exposure. MATERIAL AND METHODS: In vitro, co-cultures of human dermal fibroblasts and human dermal microvascular endothelial cells forming capillary beds were exposed to a mixture of phosphate buffered saline and either Iobitridol, Iodixanol, or one of their supplements EDTA or Trometamol for 1.5 or 5 min. Negative control co-cultures were exposed exclusively to phosphate buffered saline. Co-cultures were either directly fixed or underwent a regeneration time of 1, 3 or 7 days. An artificial intelligence software was trained for detection of labeled endothelial capillaries (CD31) on light microscope images and measurements of morphometric parameters. In vivo, we retrospectively analyzed data from patients who underwent intra-arterial administration of contrast media and for whom SCr values were available pre- and post-contrast exposition (1, 3, and 7 days following procedure). Temporal development of SCr and incidence of CA-AKI were assessed. Both exposure types were qualitatively compared. RESULTS: In vitro, Iobitridol, Iodixanol and EDTA induced a strong decrease of two morphometric parameters after 3 days of regeneration. In vivo, a significant increase of SCr and incidence of CA-AKI was observed 3 days following procedure in the post-contrast media patients. No difference was observed between groups. DISCUSSION: Two of the morphometric parameters were inversely proportional to the SCr of the patients. If the endothelial damages observed in vitro occur in vivo, it may result in renal hypoxia, inducing a loss of kidney function clinically translated into an increase of SCr. Further development of our in vitro model could allow closer replication of the internal structure of a kidney and bridge the gap between in vitro studies and their clinical findings.

Moderate Differences in Plasma Leptin in Mares Have no Effect on Either the Amino Acid or the Fatty Acid Composition of the Uterine Fluid.

Female mammalian reproductive functions are closely linked to body condition and metabolic status. Energy homeostasis is regulated by endocrine hormones such as insulin, IGF-I, leptin, and adiponectin via the hypothalamic-pituitary-adrenal axis. These metabolic hormones and their receptors are also expressed in reproductive tissues and the embryo. We investigated the relationship between circulating leptin and the fatty acid (FA) and amino acid (AA) composition of the equine uterine fluid (UF) and peripheral blood plasma (BP) by using a mass spectrometry-based approach. UF and BP were collected from ten broodmares on days 6 and 7 post ovulation, respectively. The mares were retrospectively assigned to two groups according to their BP leptin concentrations (high leptin [> 1.6 ng/mL] versus low leptin [<0.8 ng/mL]). Specific AA and FA compositions for BP and UF were found with different levels of respective metabolite abundances. The main FAs in BP were stearic, palmitic and linoleic acid. In UF, the three most abundant FAs were eicosapentaenoic, arachidonic and stearic acid. The AA profile of BP was dominated by glycine, glutamine, serine and alanine, which were likewise among the highly abundant AAs in UF. In UF, glutamic acid had by far the highest concentration. Therefore, BP leptin concentration within a physiological range does not seem to affect the specific FA nor the AA composition of the UF. The composition of the UF may therefore be mediated by local rather than by peripheral metabolic hormones.

Amino acids activate mTORC1 to release roe deer embryos from decelerated proliferation during diapause.

Embryonic diapause in mammals leads to a reversible developmental arrest. While completely halted in many species, European roe deer (Capreolus capreolus) embryos display a continuous deceleration of proliferation. During a 4-mo period, the cell doubling time is 2 to 3 wk. During this period, the preimplantation blastocyst reaches a diameter of 4 mm, after which it resumes a fast developmental pace to subsequently implant. The mechanisms regulating this notable deceleration and reacceleration upon developmental resumption are unclear. We propose that amino acids of maternal origin drive the embryonic developmental pace. A pronounced change in the abundance of uterine fluid mTORC1-activating amino acids coincided with an increase in embryonic mTORC1 activity prior to the resumption of development. Concurrently, genes related to the glycolytic and phosphate pentose pathway, the TCA cycle, and one carbon metabolism were up-regulated. Furthermore, the uterine luminal epithelial transcriptome indicated increased estradiol-17ß signaling, which likely regulates the endometrial secretions adapting to the embryonic needs. While mTORC1 was predicted to be inactive during diapause, the residual embryonic mTORC2 activity may indicate its involvement in maintaining the low yet continuous proliferation rate during diapause. Collectively, we emphasize the role of nutrient signaling in preimplantation embryo development. We propose selective mTORC1 inhibition via uterine catecholestrogens and let-7 as a mechanism regulating slow stem cell cycle progression.

Alpine and lowland grazing differentially alter the reproductive tract redox milieu and amino acid composition in cattle.

An alpine environment is unique due to pasture biodiversity, with an abundant content of natural antioxidant polyphenols. The present study investigated the effects of lowland and alpine grazing on the oviduct and uterine tissue redox status and amino acid concentrations in plasma and reproductive fluids. In the first experiment, heifers grazed on lowland (H-LOW: n = 13) and on alpine (H-ALP: n = 15) pastures. In the second experiment, heifers grazed on the same lowland (HS-LOW: n = 6) and on a different alpine (HS-ALP: n = 6) pasture. The abundance of mRNA transcripts for antioxidant enzymes in the oviduct (glutathione S-transferase alpha 2, glutathione synthetase (GSS)) and the endometrium (catalase, glutathione-disulfide reductase, GSS) was less (P <  0.05), and for glutathione peroxidase 4 in the endometrium greater (P =  0.006) in the H-LOW than in the H-ALP group. The abundance of mRNA transcript for catalase was less in the endometrium in the H-LOW than in the H-ALP (P =  0.001) group. Catalase and NAD(P)H quinone dehydrogenase 1 concentrations in the oviduct were greater in the HS-LOW than in the HS-ALP group (P <  0.05). Of 32 amino acids analysed, there were differences in concentrations in the H-LOW and H-ALP group of 13, seven and 15 in plasma, oviduct and uterine fluids, respectively (P <  0.05). Comparing the HS-LOW to the HS-ALP groups, there were 13, one and three amino acids in the plasma, oviduct and uterine fluids, respectively, that were differentially abundant (P <  0.05). The grazing systems had some effect on the redox status and amino acid patterns in reproductive tissues.

Spontaneous embryo resorption in the mouse is triggered by embryonic apoptosis followed by rapid removal via maternal sterile purulent inflammation.

BACKGROUND: In normal mammalian development a high percentage of implantations is lost by spontaneous resorption. This is a major problem in assisted reproduction and blastocyst transfer. Which embryo will be resorbed is unpredictable. Resorption is very fast, so that with conventional methods only final haemorrhagic stages are encountered. Here we describe the histology and immunohistochemistry of 23 spontaneous embryo resorptions between days 7 and 13 of murine development, which were identified by high-resolution ultrasound (US) in a previous study. RESULTS: In the early resorptions detected at day 7, the embryo proper was replaced by maternal haemorrhage and a suppurate focus of maternal neutrophils. In the decidua maternal macrophages transformed to foam cells and formed a second focus of tissue dissolution. In the late resorptions detected at day 9, the embryo underwent apoptosis without involvement of maternal cells. The apoptotic embryonic cells expressed caspase 3 and embryonic blood cells developed a macrophage like phenotype. Subsequently, the wall of the embryonic vesicle ruptured and the apoptotic embryo was aborted into the uterine lumen. Abortion was initiated by degeneration of the embryonic lacunar trophoblast and dissolution of the maternal decidua capsularis via sterile inflammation and accompanied by maternal haemorrhage, invasion of the apoptotic embryo by maternal neutrophils, and contraction rings of the uterine muscle layers. CONCLUSIONS: We conclude that spontaneous resorption starts with endogenous apoptosis of the embryo without maternal contribution. After break down of the foetal-maternal border, the apoptotic embryo is invaded by maternal neutrophils, aborted into the uterine lumen, and rapidly resorbed. We assume that the innate maternal unspecific inflammation is elicited by disintegrating apoptotic embryonic cells.

Endometrial luminal epithelial cells sense embryo elongation in the roe deer independent of interferon-tau†.

Numerous intrauterine changes take place across species during embryo development. Following fertilization in July/August, the European roe deer (Capreolus capreolus) embryo undergoes diapause until embryonic elongation in December/January. Embryonic elongation prior to implantation is a common feature among ungulates. Unlike many other ruminants, the roe deer embryo does not secrete interferon-tau (IFNτ). This provides the unique opportunity to unravel IFNτ-independent signaling pathways associated with maternal recognition of pregnancy (MRP). This study aimed at identifying the cell-type-specific endometrial gene expression changes associated with the MRP at the time of embryo elongation that are independent of IFNτ in roe deer. The messenger RNA (mRNA) expression of genes known to be involved in embryo-maternal communication in cattle, pig, sheep, and mice was analyzed in laser capture microdissected (LMD) endometrial luminal, glandular epithelial, as well as stromal cells. The mRNA transcript abundances of the estrogen (ESR1), progesterone receptor (PGR), and IFNτ-stimulated genes were lower in the luminal epithelium in the presence of an elongated embryo compared to diapause. Retinol Binding Protein-4 (RBP4), a key factor involved in placentation, was more abundant in the luminal epithelium in the presence of an elongated embryo. The progesterone receptor localization was visualized by immunohistochemistry, showing an absence in the luminal epithelium and an overall lower abundance with time and thus prolonged progesterone exposure. Our data show a developmental stage-specific mRNA expression pattern in the luminal epithelium, indicating that these cells sense the presence of an elongated embryo in an IFNτ-independent manner.

Do ovarian steroid hormones control the resumption of embryonic growth following the period of diapause in roe deer (Capreolus capreolus)?

Embryonic diapause in the European roe deer includes a period of five months from August to December in which embryonic development is extremely decelerated. Following exit from diapause, the embryo rapidly elongates and subsequently implants. In diapausing carnivores and marsupials, resumption of embryonic growth is regulated by ovarian steroid hormones. In the roe deer, the role of steroid hormones is not known to date. In the present study, progesterone (P4), estradiol-17ß (E2) and total estrogens (Etot) were determined in blood plasma and endometrium of roe deer shot in the course of regular huntings between September and December. Steroid hormone concentrations were correlated to the corresponding size of the embryo derived from ex vivo uterine flushing and to the date of sampling. The mean plasma concentrations of P4 (5.4 ± 0.2 ng/ml, mean ± SE, N = 87), E2 (24.3 ± 2.6 pg/ml, N = 86) and Etot (21.7 ± 2.6 pg/ml, N = 78) remained constant over the sampling period and were not correlated to embryonic size. Likewise, endometrial concentrations of P4 (66.1 ± 6.5 ng/g), E2 (284.0 ± 24.43 pg/g) and, Etot (440.9 ± 24.43 pg/g) showed no changes over time [corrected]. Therefore, it was concluded that ovarian steroid hormones do not play a determining role in resumption of embryonic growth following the period of diapause in the roe deer.

Bovine embryo elongation is altered due to maternal fatty acid supplementation.

The pre-implantation period is prone to embryonic losses in bovine. Embryo-maternal communication is crucial to support embryo development. Thereby, factors of the uterine fluid (UF) are of specific importance. The maternal diet can affect the UF composition. Since omega 3 fatty acids (omega 3 FA) are considered to be beneficial for reproduction, we investigated if dietary omega 3 FA affected factors in the UF related to embryo elongation. Angus heifers (n = 37) were supplemented with either 450 g of rumen-protected fish oil (omega 3 FA) or sunflower oil (omega 6 FA) for a period of 8 weeks. Following cycle synchronization and artificial insemination, the uteri were flushed post mortem to recover the embryos on day 15 of pregnancy. The UF and tissue samples of endometrium and corpus luteum (CL) were collected. Strikingly, the embryo elongation in the omega 3 group was enhanced compared to the omega 6 group. No differences were observed in uterine prostaglandins, even though the endometrial concentration of their precursor arachidonic acid was reduced in omega 3 compared to omega 6 heifers. The dietary FA neither led to differential expression of target genes in endometrium nor CL nor to a differential abundance of low-density lipoprotein cholesterol, cortisol or amino acids in the UF. Interestingly, the omega 3 group displayed a higher plasma progesterone concentration during luteal growth than the omega 6 group, possibly promoting embryo elongation. Further research should include an ovarian perspective to understand the functional link between dietary omega 3 FA and reproductive outcome.

Predicting time to full enteral nutrition in children after significant bowel resection.

PURPOSE: Parenteral nutrition (PN) contributes to considerable morbidity in children after significant bowel resection. This study evaluates the utility of clinical variables in predicting time to independence from PN. METHODS: After IRB approval, a retrospective review (1999-2012) of 71 children who were on PN for >6weeks after intestinal resection and successfully weaned was performed. Clinical characteristics were evaluated to determine the relationship to time to full enteral nutrition. P-values<0.05 were significant. RESULTS: Most children had necrotizing enterocolitis (56%), intestinal atresia (20%), or gastroschisis (11%) with a median small bowel length of 55cm (IQR, 35-92cm). The duration of PN was independent of the etiology of intestinal loss, presence of the ileocecal valve or colon, or location of anastomosis, but was strongly associated with small bowel length (<0.01) and percent of expected small bowel based on gestational age (GA) (median 50%, <0.01). In general, children who had 25-50% of their small bowel were dependent on PN for at least 2years compared to approximately 1year for those with 51-75%. CONCLUSION: The duration of PN dependence in children after major bowel resection is best predicted by remaining small bowel length and can be estimated using a linear regression model. LEVEL OF EVIDENCE: 2b.

Characterisation of major histocompatibility complex class I genes at the fetal-maternal interface of marsupials.

Major histocompatibility complex class I molecules (MHC-I) are expressed at the cell surface and are responsible for the presentation of self and non-self antigen repertoires to the immune system. Eutherian mammals express both classical and non-classical MHC-I molecules in the placenta, the latter of which are thought to modulate the maternal immune response during pregnancy. Marsupials last shared a common ancestor with eutherian mammals such as humans and mice over 160 million years ago. Since, like eutherians, they have an intra-uterine development dependent on a placenta, albeit a short-lived and less invasive one, they provide an opportunity to investigate the evolution of MHC-I expression at the fetal-maternal interface. We have characterised MHC-I mRNA expression in reproductive tissues of the tammar wallaby (Macropus eugenii) from the time of placental attachment to day 25 of the 26.5 day pregnancy. Putative classical MHC-I genes were expressed in the choriovitelline placenta, fetus, and gravid endometrium throughout the whole of this period. The MHC-I classical sequences were phylogenetically most similar to the Maeu-UC (50/100 clones) and Maeu-UA genes (7/100 clones). Expression of three non-classical MHC-I genes (Maeu-UD, Maeu-UK and Maeu-UM) were also present in placental samples. The results suggest that expression of classical and non-classical MHC-I genes in extant marsupial and eutherian mammals may have been necessary for the evolution of the ancestral therian placenta and survival of the mammalian fetus at the maternal-fetal interface.

Early detection and staging of spontaneous embryo resorption by ultrasound biomicroscopy in murine pregnancy.

BACKGROUND: Embryo resorption is a major problem in human medicine, agricultural animal production and in conservation breeding programs. Underlying mechanisms have been investigated in the well characterised mouse model. However, post mortem studies are limited by the rapid disintegration of embryonic structures. A method to reliably identify embryo resorption in alive animals has not been established yet. In our study we aim to detect embryos undergoing resorption in vivo at the earliest possible stage by ultra-high frequency ultrasound. METHODS: In a longitudinal study, we monitored 30 pregnancies of wild type C57BI/6 mice using ultra-high frequency ultrasound (30-70 MHz), so called ultrasound biomicroscopy (UBM). We compared the sonoembryology of mouse conceptuses under spontaneous resorption and neighbouring healthy conceptuses and correlated the live ultrasound data with the respective histology. RESULTS: The process of embryo resorption comprised of four stages: first, the conceptus exhibited growth retardation, second, bradycardia and pericardial edema were observed, third, further development ceased and the embryo died, and finally embryo remnants were resorbed by maternal immune cells. In early gestation (day 7 and 8), growth retardation was characterized by a small embryonic cavity. The embryo and its membranes were ill defined or did not develop at all. The echodensity of the embryonic fluid increased and within one to two days, the embryo and its cavity disappeared and was transformed into echodense tissue surrounded by fluid filled caverns. In corresponding histologic preparations, fibrinoid material interspersed with maternal granulocytes and lacunae filled with maternal blood were observed. In later stages (day 9-11) resorption prone embryos were one day behind in their development compared to their normal siblings. The space between Reichert's membrane and inner yolk sac membrane was enlarged The growth retarded embryos exhibited bradycardia and ultimately cessation of heart beat. Corresponding histology showed apoptotic cells in the embryo while the placenta was still intact. In the subsequent resorption process first the embryo and then its membranes disappeared. CONCLUSIONS: Our results provide a temporal time course of embryo resorption. With this method, animals exhibiting embryo resorption can be targeted, enabling the investigation of underlying mechanisms before the onset of total embryo disintegration.

Ultrasonography of wallaby prenatal development shows that the climb to the pouch begins in utero.

Marsupials have a functional placenta for a shorter period of time compared to that of eutherian species, and their altricial young reach the teats without any help from the mother. We have monitored the short intrauterine development of one marsupial, the tammar wallaby, with high-resolution ultrasound from reactivation of the 100-cell diapausing blastocyst to birth. The expanding blastocyst could be visualized when it had reached a diameter of 1.5 mm. From at least halfway through pregnancy, there are strong undulating movements of the endometrium that massage the expanding vesicle against the highly secretory endometrial surface. These unique movements possibly enhance exchange of uterine secretions and gases between the mother and embryo. There was a constant rate of development measured ultrasonographically from mid-gestation, regardless of when the blastocyst reactivated. Interestingly climbing movements by the fetus began in utero about 3 days before birth, mimicking those required to climb to the pouch.

Free blastocyst and implantation stages in the European brown hare: correlation between ultrasound and histological data.

The European brown hare (Lepus europaeus) is the only species with superconception, whereby the maternal reproductive tract hosts two sets of conceptuses at different developmental stages. The embryonic development of the hare has not yet been described. To understand the mechanism of superconception, we studied oviduct transport and implantation stages by embryo flushing and live high-resolution ultrasound. Ultrasound data of implantation stages is correlated with histology. In the oviduct, a mucin coat is deposited on the zona pellucida. The blastocysts enter the uterine horns on Day 5, 1 day later than in the rabbit, and directly expand approximately threefold. Spacing is accompanied by peristaltic movement of the endometrium. The mucin coat disappears and the conceptuses attach. The yolk-sac expands in the blastocoel and syncytial knobs invade the antimesometrial endometrium. Maternal blood lacunae appear in the mesometrial endometrial folds, which are subsequently invaded by the syncytiotrophoblast. The haemochorial chorioallantoic placenta forms. The yolk-sac cavity is gradually replaced by the allantois and finally by the exocoel. The different reproductive strategies of the precocial hare and the altricial rabbit are discussed. We assume that the lagomorph-specific mucin coat and the hare-specific delay of the oviduct-uterine transition are prerequisites for superconception.

Epidemiology, control and management of an EBHS outbreak in captive hares.

Here we describe an outbreak of European brown hare syndrome (EBHS) in a captive hare population. The EBHS outbreak occurred in March 2009, at the beginning of the breeding season. Overall mortality was 53% out of an original population of 61 animals. Animals between five and eleven months showed a significantly higher mortality rate than other age classes. Pregnant females either aborted their foetuses and survived or died pregnant. All foetuses (n=10) of the pregnant hares were PCR positive for EBHSV. Only one offspring born during the outbreak survived. Shortly after the outbreak, the surviving hares developed a specific anti-EBHSV titre between 1:80 and 1:2560, which dropped to 1:10-1:160 nine months later. Hares between one and three years of age developed a significantly higher titre than hares younger than one year or older than four years. Offspring born after the outbreak showed a lower titre of 1:10, indicating passive antibody transfer via placenta and milk. After two months, the titre was not detectable any longer. In December 2009, the captive population was vaccinated against EBHS virus with inactivated virus prepared from the organs of infected hares. The titres after the first vaccination ranged from 1:10 to 1:640, and after the second vaccination from 1:10 to 1:320. To estimate the effect of EBHS on reproduction, we compared the breeding seasons 2008 and 2009. Several possible sources of infection of the colony are discussed, but the definite cause could not be determined.

The long gestation of the small naked mole-rat (Heterocephalus glaber Rüppell, 1842) studied with ultrasound biomicroscopy and 3D-ultrasonography.

The naked mole-rat (Heterocephalus glaber) is one of the two known mammalian species that live in a eusocial population structure. Here we investigate the exceptionally long gestation period of 70 days observed in the mole-rat queen. The course of seven successful pregnancies in two individuals was recorded in a colony of captive naked mole-rats using ultrasound biomicroscopy (UBM) and 3D-ultrasonography. We establish a catalogue of basic reference ultrasound data for this species by describing the ultrasonographic appearance of reproductive organs, calculating growth curves to predict gestational age and defining ultrasonographic milestones to characterize pregnancy stages. Mean litter size was 10.9±2.7, of which 7.2±1.5 survived the weaning period. Mean interbirth interval was 128.8±63.0 days. The reproductive success in our colony did not differ from previously published data. In the queen the active corpora lutea had an anechoic, fluid filled centre. Using UBM, pregnancy could be detected 53 days before parturition. The period of embryonic development is assumed to last until 30 days before parturition. Embryonic resorptions were detected frequently in the queen, indicating that this might be an ordinary event in this species. We discuss the extraordinary long gestation period of this small rodent and postulate that the long gestation is beneficial to both the eusocial structure and longevity. An increased litter size, twice as large as for other rodents of similar size, seemingly compensates for the doubling of pregnancy length. We demonstrate that the lifetime reproductive effort of a naked mole-rat queen is equivalent to the mass of offspring that would be produced if all of the females of a colony would be reproducing.

Ultrasonographic monitoring of fetal development in unrestrained bonobos (Pan paniscus) at the Milwaukee County Zoo.

The bonobo, Pan paniscus, is one of the most endangered primate species. In the context of the Bonobo Species Survival Plan(®), the Milwaukee County Zoo established a successful breeding group. Although the bonobo serves as a model species for human evolution, no prenatal growth curves are available. To develop growth graphs, the animals at the Milwaukee County Zoo were trained by positive reinforcement to allow for ultrasound exams without restraint. With this method, the well being of mother and fetus were maintained and ultrasound exams could be performed frequently. The ovulation date of the four animals in the study was determined exactly so that gestational age was known for each examination. Measurements of biparietal diameter (BPD), head circumference (HC), abdominal circumference (AC), and femur length (FL) were used to create growth curves. Prenatal growth of P. paniscus was compared with the data of humans and the common chimpanzee, P. troglodytes. With respect to cranial structures, such as BPD and HC, humans have significant acceleration of growth compared with P. paniscus and P. troglodytes. In P. paniscus, growth of AC was similar to HC throughout pregnancy, whereas in humans AC only reaches the level of HC close to term. Growth rate of FL was similar in humans and the two Pan species until near day 180 post-ovulation. After that, the Pan species FL growth slowed compared with human FL. The newly developed fetal growth curves of P. paniscus will assist in monitoring prenatal development and predicting birth dates of this highly endangered species.

Ultrasonographically documented early pregnancy loss in an Asian elephant (Elephas maximus).

Early embryonic resorption or fetal loss is known to occur occasionally in captive elephants; however, this has mostly been reported anecdotally. The present study documents the case of a 24-year-old, multiparous Asian elephant cow that suffered embryonic death and resorption at around 18 weeks of gestation. From ovulation onwards, this female was sonographically examined 58 times. Blood was collected twice weekly for progestagen determination via enzyme immunoassay. On Day 42 after ovulation, a small quantity of fluid was detected in the uterine horn, which typically indicates the presence of a developing conceptus. Repeated inspections followed what appeared to be a normal pregnancy until Day 116. However, on Day 124, signs of embryonic life were absent. Progestagen concentrations started declining two weeks later, reaching baseline levels one month after embryonic death. Retrospectively, ultrasound examination revealed several abnormalities in the uterine horn. Besides an existing leiomyoma, multiple small cystic structures had formed in the endometrium at the implantation site and later in the placenta. These pathological findings were considered as possible contributors to the early pregnancy failure. PCR for endotheliotropic elephant herpes virus (EEHV) (which had occurred previously in the herd) as well as serology for other infectious organisms known to cause abortion in domestic animals did not yield any positive results. Although no definitive reason was found for this pregnancy to abort, this ultrasonographically and endocrinologically documented study of an early pregnancy loss provides important insights into the resorption process in Asian elephants.