Cardiovascular phenotype analysis in large and abnormal offspring calves in relation to assisted reproductive technology

Abstract

Large offspring syndrome/Abnormal Offspring syndrome (LOS/AOS) occurs at an increased frequency in calves derived from assisted reproductive techniques (ARTs). Specific criteria for LOS diagnosis in calves are lacking. Cardiac and vascular anomalies are common in ART derived neonates but have not been described in calves.

Calves derived from in vitro produced (IVP) embryos and referred to Cornell University for prolonged gestation were prospectively enrolled. Calves underwent phenotypic evaluation and echocardiography. Two-dimensional, M-mode, and color flow mapping echocardiography was performed to evaluate cardiac structure and function. Phenotypic vascular anomalies were recorded.

Seven calves were included in this study: four females and three males. The mean weight was 60.4 kg (range 50–74 kg). All seven calves were confirmed to have flow across the foramen ovale with distensible atrial septal membranes. A patent ductus arteriosus (PDA) was seen in 6/7 calves and could not be evaluated in one calf but auscultation was consistent with a PDA. Subjectively, all calves had evidence of right ventricular wall hypertrophy and chamber enlargement. Enlarged umbilical vessels were also noted at the time of cesarian section.

LOS/AOS calves with prolonged gestation have abnormal cardiac phenotypes including large foramen ovale with thin and abnormally distensible atrial septa.

Summary Sentence

Large and abnormal offspring calves demonstrate unique cardiac and vascular anomalies.

Introduction

Large offspring syndrome (LOS) is an epigenetic disorder naturally occurring in cattle. However, its prevalence is significantly higher among offspring produced through assisted reproductive technologies (ARTs) [1]. In vitro-produced (IVP) embryos account for over 75% of all bovine embryos transferred in North America; however, less than one-third of these embryos result in live calves [1]. This inefficiency is exacerbated by pregnancy, placental, and fetal abnormalities, common to large, and abnormal offspring syndrome (LOS/AOS). Epimutations, particularly hypomethylation, contribute to complications such as fetal overgrowth, asymmetric organ development, body wall defects, and prolonged gestation [1–3]. These issues pose significant economic and welfare challenges, including increased rates of cesarean deliveries and perinatal mortality [4].

In humans, epidemiological studies suggest that children conceived via ART exhibit differences in blood pressure, body composition, and glucose metabolism. The potential epigenetic alterations in embryos that arise directly from ART procedures can indeed result in live births; however, they may also lead to observable congenital disorders or malformations [5]. For instance, Beckwith–Wiedemann Syndrome (BWS), an epigenetic disorder characterized by a wide spectrum of clinical features including overgrowth, abdominal wall defects, macroglossia, and neonatal hypoglycemia, is more prevalent among children conceived through ART [1, 6, 7]. Importantly, cardiac defects are present in approximately 13%–20% of individuals with BWS, demonstrating a higher incidence of congenital heart disease compared to the general pediatric population [8–10]. BWS patients show an increased risk of congenital heart disease and idiopathic cardiomegaly. One study identified a moderate-sized patent ductus arteriosus (PDA) with a left-to-right shunt, emphasizing the relevance of cardiac abnormalities in BWS [11, 12]. These observations highlight the clinical significance of cardiac phenotypes in BWS and suggest that similar cardiac abnormalities could be present in LOS/AOS calves due to parallels in epigenetic disruptions between the two conditions [1].

Similar cardiovascular effects have been observed in ART mouse models, where cardiometabolic changes are linked to ART procedures rather than underlying infertility [5]. Adult male mice produced through in vitro fertilization (IVF) have demonstrated cardiovascular alterations, including impaired endothelial-dependent vasodilation, increased carotid artery stiffness, and elevated blood pressure, indicating that ART-induced vascular dysfunction may result from epigenetic changes [13]. Additionally, male mice resulting from IVF and suboptimal embryo culture conditions exhibited reduced systolic blood pressure and enlarged left hearts [14]. These findings, in conjunction with the clinical identification of calves born with severe cardiorespiratory clinical signs and difficulty transitioning from fetal to neonatal circulation, have prompted increased monitoring and evaluation at our institution. The identification of an increased incidence of cardiac abnormalities in ART-LOS calves, reinforced the need to investigate these associations further.

Given the phenotypic and epigenomic similarities between BWS in humans and LOS/AOS in cattle, these conditions highlight the potential broader implications of ART across species [15]. Thus, the aim of this study was to characterize the clinical and cardiovascular phenotypes of large offspring calves presented to a referral institution and born via cesarean section. We hypothesized that LOS/AOS calves would display unique cardiac and vascular phenotypes, potentially paralleling the cardiac abnormalities observed in BWS. Improved phenotyping of LOS/AOS calves provides valuable insights into the impact of ART on epigenetic regulation and cardiovascular health in cattle.

Methods

Animals

Any calf born derived from an IVP embryo and meeting criteria suggested by Rivera et al were included this study [16]. In short, calves were considered to meet inclusion criteria if they had a prolonged gestation, were abnormally large (>50 kilograms birthweight) and had large umbilical vasculature.

The following information was obtained for all calves enrolled in the study: gestation length, date of birth, birth weight, sex, breed, assisted reproductive technique, and outcome. Full physical examination was performed including cardiac, respiratory and gastrointestinal assessment. Assessment of the umbilicus, body wall and palate were performed to evaluate for any congenital abnormalities.

Surrogate dams of all calves were adult nulliparous Holstein heifers. All heifers were free of co-morbidities at the time of presentation.

Maternal blood samples were obtained prior to delivery for measurement of pregnancy specific protein B (PSPB). Calves born between March 2023–August 2024 were prospectively enrolled. A cohort of echocardiographic data was made available from six non-IVF Holstein calves where echocardiography was performed for an unrelated study between day 1 and day 8 of birth, were included as controls (unpublished data, courtesy of Dr Colin Schwarzwald, University of Zurich, Switzerland). These calves were all of appropriate gestational. Ethical approval was obtained for all procedures and investigation performed during this study (Cornell IACUC #2023–0116) and client consent was obtained for all calves participating in the study.

Pregnancy specific protein B

Blood from cows was collected prior to cesarian section. Serum was spun at 2000 g for 10 min and stored at −80°C until analysis. A cohort of control cows carrying artificial insemination (AI) pregnancies with subsequent normal gestation length were included for comparison. A previously validated ELISA kit was used to measure PSPB in all cows according to manufactures specification.¹

Echocardiographic assessment

Echocardiographic assessment was performed on all calves. Assessment was performed at approximately 24 h of age in all IVF calves. All images were obtained on GE Vivid e95 machine using either a 6 s or 4vC phased array transducer. Two-dimensional, motion mode (M-mode), and color flow Mapping images were obtained as previously reported [17]. In short, long axis images focused on the right ventricular outflow tract, left ventricular outflow tract, 4-chamber views of the left atrium and ventricle were obtained in addition to short axis images at the level of the chordae tendinea, mitral valve, and aortic valve. M-mode images were obtained at the level of the chordae, mitral valve, and aortic valve. Special views were obtained to evaluate the foramen ovale and ductus arteriosus. Color flow Mapping was used to evaluate all valves for regurgitation in both short and long axis as well as in the region of the foramen ovale and ductus arteriosus. Regurgitation was considered mild if the regurgitant jet was less than 1/3 of the valve length and extended less than 1/3 of the receiving chamber area but persisted through the entire portion of the expected cardiac cycle (systole- mitral and tricuspid regurgitation; diastoleaortic and pulmonary regurgitation). Regurgitation was considered moderate if it was between 1/3 and 2/3 of the valve length and extended between 1/3 and 2/3 of the receiving chamber area. Regurgitation was considered severe if the regurgitant orifice was greater than 2/3 of the valve length and extended more than 2/3 of the receiving chamber area. When possible, continuous wave Doppler was used to evaluate the direction and velocity of regurgitation or shunting. Images or cine loops were stored in raw format and uploaded to EchoPAC (EchoPAC Software v3.1.3, GE Medical Systems) for measurement by a single observer. Measurements were performed as previously described and compared to the unpublished data and previous publications of age matched control calves when possible [18–21].

Statistics

Data was checked for normality using Shapiro–Wilks test. Data was found to be normally distributed and is presented as mean and standard deviation. Descriptive statistics were performed on echocardiographic variables. Results are reported as mean and standard deviation of all echocardiographic measurements. All variables were compared between LOS/AOS and control calves using an unpaired t test with a welch correction for multiple comparisons. P < 0.05 are considered significant.

Results

Animals and physical examination

Seven LOS/AOS calves were included in this study for analysis. The mean gestation length was 290 days (SD 6 days). There were four females and three males. The mean weight was 60.4 kg (SD 9.1).

All calves had abnormalities noted on physical examination at the time of birth. Four out of seven calves had crackles bilaterally on thoracic auscultation and 3/7 had harsh bronchovesicular sounds. One calf was oxygen dependent. The abnormal thoracic auscultation made cardiovascular assessment challenging in all calves. A continuous left basilar murmur was heard in 6/7 calves consistent with a PDA. The calf with the ductus arteriosus was unable to be assessed on echocardiogram but had a murmur consistent with a PDA. Four of the LOS/AOS calves survived to discharge, while three calves were euthanized due to severe cardiorespiratory disease not responsive to medical treatments. The single non- LOS/AOS IVF calf and all control calves survived and appeared clinically normal at birth. Signalment and descriptive data from the LOS/AOS and control calves can be found in Table 1.

Table 1.

Gestation length, signalment, physical exam findings are reported for all seven LOS calves. Valvular regurgitation, foramen ovale flow, and ductus arteriosus flow identified on echocardiographic examination are reported in each calf in part B

A)	Calf 1	Calf 2	Calf 3	Calf 4	Calf 5	Calf 6	Calf 7
Gestation length (days)	295	296	Unknown	284	285	Unknown	292
Sex	Male	Female	Male	Female	Female	Male	Female
Breed	Holstein	Holstein	Brown Swiss	Holstein	Holstein	Holstein	Holstein
Weight (kg)	74	50	56	51	63	70	59
Outcome	Euthanized	Discharged	Euthanized	Discharged	Discharged	Euthanized	Discharged
Heart rate (bpm)	130	140	120	96	176	120	120
Respiratory rate (bpm)	60	92	48	40	52	48	100
Thoracic auscultation	Crackles & wheezes bilaterally	Crackles bilateral	Crackles bilateral, oxygen dependent	Harsh bronchovesicular sounds bilaterally	Harsh bronchovesicular sounds bilaterally	Harsh bronchovesicular sounds bilaterally	Crackles bilaterally
Cardiac auscultation	Continuous murmur over the left heart base	no murmur reported	Continuous murmur over the left heart base	Continuous murmur over the left heart base	Continuous murmur over the left heart base	Continuous murmur over the left heart base	Continuous murmur over the left heart base
B)
Echocardiographic Findings
Mitral valve regurgitation	None	Trivial	Moderate	None	Mild	Mild	None
Tricuspid valve regurgitation	Mild to moderate	Trivial	Moderate	Mild	Mild	Moderate to severe	Moderate
Aortic valve regurgitation	None	Mild to moderate	Mild to moderate	None	Mild	None	None
Pulmonary valve regurgitation	Moderate	Not evaluated	Mild	Moderate	Mild	Moderate	Moderate
PFO flow	Left to right	Left to right	Left to right	Left to right	Left to right	Left to right	Left to right
PDA flow	Not evaluated	Left to right	Left to right	Left to right	Left to right	Left to right	Left to right

Enlarged umbilical vessels with increased hemorrhage at the time the umbilicus was cut were noted in all LOS/AOS calves, prompting umbilical clamps or surgical ligation to be placed in certain instances (Figure 1).

Figure 1.

Representative image of enlarged umbilical structures noted in LOS/AOS calves. Enlarged umbilicus immediately post cesarian section and clamping (A). Trimmed section of umbilical cord at the level of the calf’s umbilicus immediately following cesarian section in LOS calf (B). Healthy c ontrol IVF calf (C).

Pregnancy specific protein B

A convenience sample of seven control and 10 IVF cows included in this analysis. No significant difference was noted in PSPB between the IVF and the AI control calves (P = 0.8). The prolonged gestation IVF cows had a mean ± S.D. PSPB of 1107 ± 479.3 pg/ml and the AI controls had a median of 1159 ± 343.6 pg/ml. The results of the PSPB are presented in Figure 2.

Figure 2.

Figure 1 Box-plot of PSPB concentrations using Sandwich ELISA. No significant difference in PSPB levels between prolonged gestation IVF pregnancies (mean: 1107 pg/ml, range: 615.9–1344 pg/ml) and control pregnancies (median: 1159 pg/ml, Range: 869–1863 pg/ml), P = 0.8 (Welch t test).

Echocardiographic examination

All seven LOS/AOS calves received complete echocardiographic assessment. All calves were confirmed to have flow across the foramen ovale in a left to right direction and 6/7 were confirmed to have a PDA (Figs. 3 and 4). Valvular regurgitation was noted in all calves; 4/7 calves had moderate or severe tricuspid valve regurgitation, 4/7 calves had moderate pulmonary valve regurgitation, 2/7 had moderate aortic valve regurgitation, and 1/7 had moderate mitral valve regurgitation. LOS/AOS calves had evidence of right ventricular chamber dilation on 2D measurements compared to control calves (mean difference: 0.9 cm, P = 0.010). Additionally, enlarged moderator bands were noted in 5/7 calves with subjective basilar insertion on the interventricular septum toward the tricuspid valve compared to control calves. Compared to control calves, LOS/AOS calves had increased body weights (mean difference: 11.4 kg, P = 0.045) and elevated heart rates (mean difference: 18 bpm, P = 0.035). The echo results are summarized in Table 2.

Figure 3.

Representative example of a patent ductus arteriosus in one calf. This image is obtained from the left parasternal long axis angled dorso-caudally to focus on the pulmonary artery and ductus arteriosus. The left panel shows the pulmonary artery (PA), aorta (A), and ductus arteriosus (red arrow). The right panel is the same image with overlayed color flow mapping. The ductus is very large with significant turbulent flow (green on the color flow mapping) traversing from the aorta into the pulmonary artery.

Figure 4.

Large foramen ovale in LOS calf. Representative images of the large and distensible foramen ovale membrane in a single LOS calf. The membrane can be seen bowing to both the right and the left (red arrows) atria throughout the cardiac cycle. RA- right atrium, LA- left atrium.

Table 2.

Echocardiographic variables in LOS Calves. Control population consists of six Holstein calves between 1 and 8 days of age (mean = 2 days, unpublished data, courtesy of University of Zurich). The P value column denotes significant difference between the control calves and the LOS calves. A single IVF but non LOS calf is included for comparison but is not included in statistical analysis. Explanation of abbreviations can be found in the supplementary material.

Variable	Control holstein calves (mean ± SD) n = 6	IVF non-LOS (mean) n = 1	LOS calves (mean ± SD) n = 7	P value
Heart rate	120 ± 11	123	138 ± 16	0.035
Weight (kg)	49 ± 9	36	60.4 ± 9.1	0.045
Left atrial variables
LADmax (cm)	5.1 ± 0.5	3.9	5.4 ± 0.5	0.285
LAAmax (cm2)	19.4 ± 4.9	10.9	22 ± 4.7	0.353
LAsxAmax (cm2)	22.5 ± 5.6	14.9	23 ± 8.9	0.905
Left ventricular variables
%EF	76.4 ± 6.2	68.1	78 ± 5.4	0.633
LVIVd (ml)	67.8 ± 24.3	43.7	83 ± 27	0.308
LVIVs (ml)	16.6 ± 9.3	14.0	18 ± 7.1	0.770
SV (ml)	51.4 ± 15.2	29.8	65 ± 21	0.205
CO (L/min)	6.2 ± 1.6	6.1	8.9 ± 0.2	0.009
Cardiac index (L/min/m2)	4.9 ± 0.9		6.8 ± 1.7	0.031
%FS	33.0 ± 2.5	40.6	50 ± 6.0	<0.001
LVIDd (cm)	3.8 ± 0.4	3.9	4.4 ± 0.4	0.019
LVIDs (cm)	2.5 ± 0.2	2.3	2.2 ± 0.4	0.141
IVSd (cm)	1.5 ± 0.3	1.1	1.7 ± 0.3	0.226
IVSs (cm)	1.6 ± 0.3	1.8	2.0 ± 0.3	0.028
LVPWd (cm)	0.8 ± 0.0	0.8	1.2 ± 0.3	0.008
LVPWs (cm)	1.6 ± 0.1	1.3	2.0 ± 0.2	<0.001
MWT (cm)	1.1 ± 0.2	1.0	1.5 ± 0.2	0.006
RWT	0.6 ± 0.1	0.5	0.7 ± 0.2	0.338
Right ventricular variables
RVIDd (cm)	2.3 ± 1.4	1.3	1.9 ± 0.2	0.518
RVIDs (cm)	1.3 ± 1.1	0.5	1.6 ± 0.5	0.555
RVID (2D) (cm)	2.4 ± 0.5		3.3 ± 0.5	0.010
RVFWd (2D) (cm)	1.0 ± 0.2		1.2 ± 0.2	0.107
IVSd (2D) (cm)	1.0 ± 0.2		1.2 ± 0.2	0.189
RVID (2D)/ RVPWd	2.5 ± 0.9		2.9 ± 0.7	0.423
RWT-RV	0.9 ± 0.2		0.7 ± 0.2	0.239
Great vessels
AoDed (cm)	2.7 ± 0.3	2.5	2.9 ± 0.3	0.257
PADed (cm)	2.4 ± 0.2	1.8	3.4 ± 0.5	0.002
PAsxDed	1.8 ± 0.5	2.2	2.4 ± 0.4	0.1743
PADed/AoDed	0.9 ± 0.1	0.7	1.2 ± 0.2	0.006
PAsxDed/AoDed	0.7 ± 0.1	0.9	0.8 ± 0.1	0.0006
AADs (cm)	2.1 ± 0.1	1.9	2.3 ± 0.2	0.0446
AosxA (cm2)	6.1 ± 1.7	4.8	6.8 ± 1.3	0.430
AoD/PAD ratio	1.1 ± 0.2	1.4	0.9 ± 0.3	0.074
Other measurements
PDA diameter (cm)			0.8 ± 0.1
Foramen ovale diameter (cm)		0.8	1.7 ± 0.5

Compared to control calves, LOS calves had larger pulmonary arteries (mean difference: 1.0 cm, P = 0.002). The pulmonary artery was enlarged relative to the aorta in both longitudinal (mean difference: 0.2 cm, P = 0.006) and short axis (mean difference: 0.3 cm, P < 0.001). Compared to control calves LOS calves had a higher cardiac index (mean difference: 1.9, P = 0.031). A single IVF but non-LOS calf is included in Table 2 for comparison but is not included in statistical analysis.

Discussion

This investigation provides new insight into the cardiac phenotype of LOS/AOS calves. While cardiac differences have been described in other species, the cardiac and vascular effects of LOS/AOS in calves have not been described to the authors knowledge [8–10]. Improved phenotyping and identification of biomarkers in calves is critical to develop scoring systems and biomarkers, similar to those that are established in human medicine [6].

The differences in body weight of these calves compared to controls, demonstrate that they are larger in size compared to their age matched controls. The large amount of valvular regurgitation in these calves was an unexpected finding. Aortic valve regurgitation is very abnormal in any neonatal species, and could reflect blood pressure or embryological development differences [22, 23]. Additionally, tricuspid, aortic, and mitral valve dysplasia are possible in these calves and could be due to abnormal blood flow during development, or directly relate to the underlying epigenomic mechanisms of LOS/AOS [22]. While these regurgitations were noted on echo, specific murmurs were not detected on physical highlighting that these abnormalities have likely been previously overlooked. Additionally, the large and basilar insertion of the moderator band in the right ventricle could contribute to tricuspid valve dysfunction and cause some degree of right ventricular inflow obstruction.

The cardiac measurements obtained in this study show significant differences compared to healthy age matched control calves. Although many can be attributed to bodyweight (and therefore cardiac size) differences in the two cohorts of calves, the larger birth weight associated with increased gestational length beyond the typical 285-day average observed in these calves is a common finding in LOS/AOS calves [16]. The increase in cardiac output in LOS calves can partly be explained by the elevated heart rate of this cohort and larger ventricle size proportional to body weight. The increased cardiac index, however, accounts for differences in body weight, and indicates that these calves have a larger circulating blood volume for their given body weight. Pulmonary artery enlargement in LOS/AOS calves compared to control calves is an important finding. While calf size could contribute to this difference, the pulmonary artery is enlarged relative to the aorta in both longitudinal and short axis images suggesting that the pulmonary artery is truly enlarged in LOS/AOS calves. This enlargement could also be partly due to the large PDA noted in these calves causing over circulation of the pulmonary vasculature. While the ductus arteriosus is expected to still be open at 24 h of age, the large size of the ductus in these calves (nearly 1/3 the size of their aortic diameter), appears inappropriate and may impair the spontaneous closure of the ductus over time. Additionally, the presence of abnormal lung sounds in all calves suggest that there is active pulmonary pathology. Invasive blood pressures within the right heart and pulmonary artery as well as additional measurement of shunt and valvular regurgitation velocities could help to better determine the cause and relevance of the pulmonary artery dilation.

Flow across the foramen ovale is considered abnormal in all calves after birth. In a normal calf, the foramen between the atria should functionally close when increased left cardiac pressure and decreased right cardiac pressure occurs associated with decreasing pulmonary vascular resistance and termination of fetal circulation. In cases of a patent foramen ovale, the membranous septum primum and septum secundum that make up the foramen fail to seal the opening between the right and left atria, and blood continues to flow between the atrial chambers, typically in a right to left direction [24]. This can be due to an abnormally distensible membrane, pathologic shortening of the septum primum or secundum membrane or with abnormally high left atrial pressures causing blood to shunt left to right across the foramen ovale or due to a pathologically enlarged gap between the atria (atrial septal defect) that occurs during embryological development. Many of these LOS/AOS calves appear to have an area between the atria that is concerning for an ASD or abnormally stretched foramen ovale, with a large defect and an easily distensible membranous septum that doesn’t always appear to close the gap [25]. While the exact mechanism of this abnormality remains unknown, it is important to note that this pathologic change allows blood flow to continue to cross between the atria, supporting a diagnosis of persistent fetal circulation in these calves. All calves had flow from left to right across the foramen ovale, suggesting either the presence of an atrial septal defect or reversed PFO flow through a stretched foramen. Further work is needed to better understand the exact embryologic mechanism of these atrial septal abnormalities.

In LOS/AOS calves with delayed transition from fetal to neonatal life, cardiac evaluation should be performed to screen for persistent fetal circulation, clinically relevant valvular regurgitation, evidence of pulmonary hypertension, structural defects or complex congenital abnormalities that could have life or production limiting consequences [17]. Early recognition of hemodynamically relevant cardiac abnormalities will help inform decision making, as management of these calves can be labor intensive and expensive.

In addition, long term follow-up of calves with cardiac abnormalities detected on echocardiography is essential to understand the hemodynamic relevance of the valvular regurgitation and intra-cardiac shunts, and to determine if these shunts are able to close eventually or remain patent. It is also important to understand if there is a hereditary component to any of these structural cardiac abnormalities.

Placental abnormalities are well described in human IVF pregnancies [26]. The enlarged umbilical structures in LOS/AOS calves suggest either a disproportionate vascular development pattern or large volume of blood circulating across the umbilical vessels throughout pregnancy. This could be a consequence of asymmetric vascular organ enlargement, increased placental blood volume, placental development or some combination of the above [27]. Previous reports have indicated that IVF pregnancies with prolonged gestation are associated with lower maternal concentrations of PSPB [28]. This suggests that there are inherent differences in the placental trajectory of IVF pregnancies with prolonged gestation. Our cohort failed to demonstrate this same clear difference in prolonged gestation IVF pregnancies. Further work investigation into the placental anatomy, signaling and function is needed for bovine IVF pregnancies.

This study has several limitations. The descriptive nature and small sample size of this study limits the scope of investigation. Calves that were hospitalized at a referral institution and thus some bias toward more severely affected calves could exist. Additionally, an exact age and sex matched control group from a similar population of calves could have improved the ability to interpret echocardiographic findings.

In conclusion, this investigation describes the cardiac and vascular phenotypes of LOS/AOS calves in a hospital population. The cohort demonstrates the importance of cardiovascular investigation in clinical calves derived from ART and demonstrating abnormal cardiopulmonary signs at birth. Furthermore, the resulting cardiac phenotypes of LOS/AOS calves highlight the need for further investigation due to their economic and welfare impact on the cattle industry. Future directions include investigation into vascular anomalies and placental blood flow in LOS/AOS placentas long term follow-up of calves with cardiac anomalies, and the development of an LOS/AOS scoring system in calves are needed.

Supplementary Material

Supplementary data are available at BIOLRE online.

Data availability

Data are available upon reasonable request.