Fetal Sex Does Not Impact Placental Blood Flow or Placental Amino Acid Transfer in Late Gestation Pregnant Sheep With or Without Placental Insufficiency

Abstract

Pregnant sheep have been used to model complications of human pregnancies including placental insufficiency and intrauterine growth restriction. Some of the hallmarks of placental insufficiency are slower uterine and umbilical blood flow rates, impaired placental transport of oxygen and amino acids, and lower fetal arterial concentrations of anabolic growth factors. An impact of fetal sex on these outcomes has not been identified in either human or sheep pregnancies. This is likely because most studies measuring these outcomes have used small numbers of subjects or animals. We undertook a secondary analysis of previously published data generated by our laboratory in late-gestation (gestational age of 133 ± 0 days gestational age) control sheep (n = 29 male fetuses; n = 26 female fetuses; n = 3 sex not recorded) and sheep exposed to elevated ambient temperatures to cause experimental placental insufficiency (n = 23 male fetuses; n = 17 female fetuses; n = 1 sex not recorded). The primary goal was to determine how fetal sex modifies the effect of the experimental insult on outcomes related to placental blood flow, amino acid and oxygen transport, and fetal hormones. Of the 112 outcomes measured, we only found an interaction between fetal sex and experimental insult for the uterine uptake rates of isoleucine, phenylalanine, and arginine. Additionally, most outcomes measured did not show a difference based on fetal sex when adjusting for the impact of placental insufficiency. Exceptions included fetal norepinephrine and cortisol concentrations, which were higher in female compared to male fetuses. For the parameters measured in the current analysis, the impact of fetal sex was not widespread.

Introduction

A healthy placenta is necessary for a successful pregnancy. There are myriad functions carried out by the placenta, and abnormalities in many of these underlie the most common obstetrical complications. Among these, we focus on placental insufficiency resulting in intrauterine growth restriction (IUGR) of the fetus [1]. Placental insufficiency is defined as a failure of the placenta to meet the nutritional and oxygen requirements needed by the fetus to reach its genetic growth potential [2].

Transport of maternal amino acids to the fetus is one of the key placental functions that allows for appropriate fetal growth and development. Pregnancies complicated by IUGR demonstrate reduced placental transfer of essential amino acids [3]. This has been demonstrated in vivo using a stable isotope of leucine to show that restricted placental leucine transport correlates with the severity of IUGR as estimated by Doppler blood flow patterns in the fetal circulation. The greater the severity of placental insufficiency, the greater the decrement of leucine transport [4]. Impaired placental leucine transport and impaired activity of the leucine transporters have been demonstrated in vitro using isolated human tissue [5–8].

Multiple animal models also demonstrate the importance of placental amino acid transport in the pathogenesis of IUGR. In nutrient-restricted baboons, reductions in placental amino acid transporters precede reductions in fetal weight [9]. In rats, IUGR can be modeled with a maternal isocaloric low-protein diet [10]. In fact, similar to the nutrient-restricted baboon, reductions in placental amino acid transport precede slower fetal growth rates in maternal isocaloric low-protein diet models of IUGR [11]. We have used pregnant sheep to not only model placental insufficiency and IUGR [12], but also to quantify the rate of transport of individual amino acids from the maternal circulation into the placenta and from the placenta into the fetal circulation [13–15]. These are termed uterine uptake and umbilical uptake rates, respectively. We can accomplish this in pregnant sheep by surgically placing sampling and infusion vascular catheters on both sides of the placenta, which will allow us to sample the blood entering and exiting the placenta in both the maternal and fetal circulations. When combined with the steady-state transplacental tritiated water diffusion technique to measure uterine and umbilical blood flow, we can apply the Fick principle to calculate uterine and umbilical uptake of oxygen and amino acids in the non-anesthetized, non-stressed condition for both the fetus and the ewe. Using this technique, we have consistently demonstrated lower umbilical amino acid uptake per kilogram of fetus in pregnancies complicated by placental insufficiency [16–19].

The impact of fetal sex on placental development and function is important to consider. Differences in certain aspects of placental function and morphology based on sex of the fetus have been demonstrated in human and animal pregnancies [20–22]. However, differences in placental blood flow and amino acid transport based on fetal sex have not been identified. Whether this is because there is no difference based on fetal sex or because the numbers of study subjects have been insufficient, thus resulting in a type II error, is unknown. The impact of fetal sex on these placental functions is relevant because in human pregnancies, male fetuses are slightly heavier at the end of gestation. Data supporting this come from large studies [23, 24]. Studies without these large sample sizes typically do not show a size difference between male and female newborn weights because the variability of birth weight obscures any differences based on sex. The same is true of sheep pregnancies. With large enough samples, male lambs at birth have been shown to be heavier than female lambs [25, 26], though most studies using late-gestation or newborn sheep fail to show this difference due to small sample sizes. Similarly, while we have consistently tested for an effect of fetal sex on placental blood flows and amino acid transport, typically the numbers of animals in any given study are too small to detect potentially subtle but consistent differences based on sex of the fetus [13, 14, 17].

Therefore, the primary goal of this study was to combine data from previously published and ongoing studies in normal pregnant sheep and in pregnant sheep with experimentally induced placental insufficiency and fetal growth restriction to specifically interrogate the interaction between fetal sex and IUGR on fetal and placental weight, placental blood flow, oxygen and amino acid uptake rates, and fetal hormones that regulate growth and development. In this large cohort of animals, we also examined the relationships between amino acid uptake rates and circulating anabolic and catabolic hormone concentrations with fetal weight.

Methods

Ethical Approval

Study protocols were approved by the Institutional Animal Care and Use Committee at the University of Colorado Anschutz Medical Campus and are in compliance with guide-lines from AAALAC. Experiments were performed at the Perinatal Research Center on the University of Colorado Anschutz Medical Campus.

Animal Care

Pregnant Columbia-Rambouillet mixed-breed sheep obtained from Nebeker Ranch (Lancaster, CA) were used in this study. Data from many of these animals have been reported in previous manuscripts [13, 14, 17, 27–29]. Animals from these published manuscripts are included in the current study if they meet the following criteria: (1) singleton pregnancy, (2) no chronic experimental infusions other than a vehicle control infusion, and (3) underwent baseline steady-state measurements of uterine and/or umbilical blood flows with the transplacental tritiated water diffusion technique, oxygen uptake rates, and amino acid uptake rates. Other recently studied animals are included if they met the same criteria. Some animals were housed in environmental chambers and exposed to elevated ambient temperatures (40 °C for 12 h; 35 °C for 12 h) and 40% humidity from approximately 35–40 days gestation age (dga, term = 147 dga) to approximately 110–120 dga to induce placental insufficiency and IUGR [12].

Surgical Procedure and Animal Numbers

The surgical procedure was as described in previous publications [13, 14]. At approximately 118–125 dga, animals underwent surgical placement of fetal and maternal catheters. The uterus was exteriorized via midventral laparotomy, and the fetus was exposed through an incision in the uterine wall. Fetuses were instrumented with indwelling catheters in their descending aorta (representing umbilical arterial blood), femoral vein, and umbilical vein. Maternal femoral artery (representing uterine artery blood), femoral vein, and uterine vein were also catheterized. All catheters were tunneled subcutaneously to the maternal flank, where they were exteriorized into a pouch. Animals were allowed to recover a minimum of 5 days prior to any metabolic study.

Ninety-nine animals were included in this study. Fifty-eight of these were normally growing control fetuses (n = 29 male fetuses; n = 26 female fetuses; n = 3 sex not recorded; two of these three animals did not have uterine blood flow or uterine uptake rates measured), and forty-one of these were IUGR fetuses (n = 23 male fetuses; n = 17 female fetuses; n = 1 sex not recorded; this animal did not have uterine blood flow or uterine uptake rates measured). Not all animals had both an umbilical vein catheter and a uterine vein catheter placed, and in some animals, these catheters were placed but were not functional at the time of their metabolic study. Thirty-one animals had both umbilical and uterine blood flows and uptake rates measured. Sixty-three animals had just umbilical blood flow and uptake rates measured, though in three of these animals, amino acid concentrations were not measured and umbilical amino acid uptake rates could not be calculated. Four animals had just uterine blood flow and uptake rates measured. Fetal arterial plasma insulin was measured in all animals. Cortisol was not measured in one animal. Norepinephrine was not measured in two animals. Insulin-like growth factor 1 (IGF-1) was not measured in fourteen animals. Glucagon was not measured in forty-two of the animals.

Transplacental Tritiated Water Diffusion Technique to Measure Uterine and Umbilical Blood Flow and Nutrient Uptake Rates

The transplacental tritiated water diffusion technique and Fick principle were used to measure uterine and umbilical blood flows and nutrient and oxygen uptake rates as previously described [13]. This technique is based on the principle that placental uptake of the inert molecule, tritiated water, is flow-limited and that the uptake of tritiated water is equal to its infusion rate at steady state. The Fick equation (Uptake = ([C]_in − [C]_out) × Q, where [C]_in and [C]_out refer to the concentration of tritiated water entering the placental and leaving the placenta, respectively, and Q refers to blood flow) can then be rearranged to calculate umbilical blood flow. [C]_in and [C]_out can be reversed to calculate uterine blood flow. Once uterine and umbilical and blood flows are calculated, the Fick equation can then be used to calculate uterine and umbilical uptake rates of other nutrients and oxygen. Baseline samples (draw 0) were collected from the maternal femoral artery, uterine vein, umbilical vein, and fetal descending aorta simultaneously. Then, a 3-mL bolus of ³H₂O (15 μCi/mL) was infused into the fetal femoral vein, and isotopic steady state was reached by a continuous infusion at 3 mL/h (15 μCi/mL) for 90 min. Four samples (draws 1–4) were then collected from the four catheters simultaneously at 15- to 20-min intervals for analysis of oxygen, amino acid concentrations, ³H₂O, and hormone concentrations. Hematocrit was determined by the ABL 825 Blood Gas Analyzer which allowed for calculation of plasma flow from blood flow.

Ewes and fetuses were euthanized within 24 h of the metabolic study by a lethal dose of sodium pentobarbital (390 mg/mL, Fatal Plus; Vortech Pharmaceuticals, Dear-born, MI). Placentomes were trimmed from the endometrium and weighed. The fetus was weighed, and sex was recorded.

Biochemical Assays

The partial pressure of blood oxygen, blood hemoglobin concentrations, and hemoglobin-oxygen saturations were measured by the ABL 825 Blood Gas Analyzer, and oxygen content of the blood was then calculated by this device [13]. Plasma amino acid and norepinephrine concentrations were measured by high-performance liquid chromatography; plasma insulin, IGF-1, and cortisol concentrations were measured by enzyme-linked immunosorbent assay; and plasma glucagon concentrations were measured by radio-immunoassay [13]. Intra-assay, inter-assay, and sensitivity of the immunoassays were as follows: insulin, 5.6%, 4.7%, and 0.14 ng/ml; norepinephrine, 9.2%, 9.0%, and 170 pg/ml; cortisol, 4.6%, 5.8%, and 1.0 ng/ml; glucagon, 4.8%, 11.7%, and 18.5 pg/ml; and IGF-1, 3.1%, 5.6%, and 0.09 ng/ml.

Statistical Analysis

The effect of sex on the association between parameters and experimental placental insufficiency–induced IUGR was tested using linear models with an interaction term. Pearson’s correlation was used to assess the correlation between umbilical uptake rates of various amino acids or fetal plasma arterial hormone concentrations and fetal weight. Linear models, with an interaction term, were used to assess the effect of group (control vs. IUGR) on the association between hormones and fetal weight. All variables were assessed for normality using histograms and log-transformed as appropriate. R version 4.0.2 software (R Foundation for Statistical Computing, Vienna, Austria; http://www.R-project.org/), Prism version 9.0.0, and Excel for Microsoft 365 were used for analysis. One animal was not included when determining the correlation between cortisol and norepinephrine with fetal weight. This was an IUGR fetus that weighed 1.734 kg and had a plasma arterial cortisol concentration of 709 ng/mL and a plasma arterial norepinephrine concentration of 17,111 pg/mL. Means and 95% confidence intervals of all parameters are provided in Table 1. For further analysis of fetal weight, we performed an analysis restricted to just control animals and report descriptive statistics with mean and 95% confidence intervals for males and females and comparative statistics using a two-sample t test.

Table 1.

Impact of fetal sex and experimental IUGR

Variable	Means (95% confidence interval)				P values
	Female		Male		IUGR vs. control	Male vs. female	Interaction
	Control	IUGR	Control	IUGR
Placental weight (g)	332.16 (304.92, 359.41)	168.91 (137.79, 200.03)	357.32 (331.15, 383.48)	194.06 (165.20, 222.93)	< 0.0001	0.1280	0.4481
Fetal weight (g)	3225.85 (3045.7, 3406)	1955.76 (1750.22, 2161.29)	3326.58 (3153.54, 3499.62)	2056.48 (1868.49, 2244.48)	< 0.0001	0.3536	0.3982
Fetal arterial blood oxygen (mmol/L)	2.75 (2.49, 3.01)	1.85 (1.56, 2.14)	2.88 (2.64, 3.12)	1.98 (1.71, 2.25)	< 0.0001	0.4066	0.6451
Fetal arterial plasma hormones
IGF-1 (ng/mL)	106.35 (95.34, 117.37)	56.32 (43.45, 69.19)	112.89 (102.38, 123.41)	62.86 (50.87, 74.85)	< 0.0001	0.3287	0.1292
Insulin (ng/mL)*	0.40 (0.33, 0.48)	0.17 (0.14, 0.22)	0.38 (0.32, 0.46)	0.17 (0.14, 0.20)	< 0.0001	0.7105	0.1287
Norepinephrine (pg/mL)*	568.48 (447.49, 722.18)	1549.87 (1178.73, 2037.87)	405.05 (321.90, 509.67)	1104.29 (853.61, 1428.58)	< 0.0001	0.0209	0.9385
Cortisol (ng/mL)*	15.07 (10.95, 20.74)	32.46 (22.54, 46.73)	8.99 (6.61, 12.21)	19.35 (13.80, 27.13)	0.0002	0.0083	0.5691
Glucagon (pg/mL)*	46.22 (35.89, 59.51)	68.56 (47.86, 98.23)	43.04 (32.68, 56.69)	63.85 (46.26, 88.14)	0.0331	0.6742	0.2352
Blood and plasma flows (mL/min)
Umbilical blood flow	601.57 (549.17, 653.97)	258.58 (199.35, 317.81)	638.68 (587.74, 689.63)	295.69 (240.06, 351.33)	< 0.0001	0.2421	0.2695
Umbilical plasma flow	402.62 (365.42, 439.83)	163.15 (121.09, 205.21)	428.50 (392.33, 464.67)	189.03 (149.52, 228.53)	< 0.0001	0.2507	0.3360
Uterine blood flow	1706.92 (1460.57, 1953.28)	988.96 (581.94, 1395.99)	1854.02 (1548.25, 2159.79)	1136.06 (799.64, 1472.48)	0.0009	0.4139	0.5819
Uterine plasma flow	1229.02 (1042.72, 1415.33)	638.95 (331.14, 946.77)	1362.69 (1131.45, 1593.93)	772.62 (518.20, 1027.04)	0.0004	0.3274	0.6739
Blood and plasma flows relative to fetal weight (mL/min/kg)
Umbilical blood flow*	180.58 (167.23, 195)	130.19 (119.36, 142.00)	184.58 (171.30, 198.88)	133.07 (122.65, 144.38)	< 0.0001	0.6370	0.4221
Umbilical plasma flow*	119.60 (109.54, 130.58)	82.05 (74.3, 90.62)	123.47 (113.36, 134.48)	84.71 (77.17, 93)	< 0.0001	0.5477	0.6375
Blood and plasma flows relative to placental weight (mL/min/kg)
Uterine blood flow*	5.34 (4.73, 6.04)	4.99 (4.05, 6.15)	5.33 (4.58, 6.20)	4.98 (4.13, 6.01)	0.5049	0.9797	0.1476
Uterine plasma flow*	3.81 (3.35, 4.35)	3.26 (2.61, 4.08)	3.91 (3.32, 4.60)	3.35 (2.73, 4.09)	0.1628	0.7920	0.2096
Umbilical oxygen and amino acid uptake rates (μmol/min)
Oxygen	1153.40 (1072.34, 1234.46)	597.81 (506.19, 689.44)	1226.56 (1147.76, 1305.37)	670.98 (584.91, 757.04)	< 0.0001	0.1370	0.3361
Alanine	9.71 (8.33, 11.09)	4.47 (2.87, 6.06)	10.87 (9.51, 12.23)	5.63 (4.13, 7.12)	< 0.0001	0.1712	0.6744
Arginine	8.17 (7.18, 9.15)	3.37 (2.23, 4.50)	9.46 (8.49, 10.43)	4.65 (3.59, 5.72)	< 0.0001	0.0343	0.3060
Asparagine	3.57 (2.54, 4.6)	2.94 (1.76, 4.13)	2.60 (1.59, 3.61)	1.98 (0.86, 3.09)	0.3278	0.1251	0.4027
Aspartate	− 0.20 (− 0.61, 0.21)	− 0.17 (− 0.64, 0.30)	− 0.07 (− 0.47, 0.33)	− 0.04 (− 0.48, 0.40)	0.8973	0.6138	0.7178
Citrulline	− 0.08 (− 0.94, 0.79)	0.75 (− 0.25, 1.75)	− 0.26 (− 1.12, 0.59)	0.56 (− 0.37, 1.50)	0.1238	0.7240	0.1815
Cystine	0.27 (0.04, 0.51)	0.13 (− 0.15, 0.4)	0.49 (0.26, 0.73)	0.35 (0.09, 0.60)	0.3141	0.1323	0.6438
Glutamate	− 10.58 (− 12, − 9.17)	− 3.80 (− 5.43, − 2.17)	− 11.67 (− 13.07, − 10.27)	− 4.89 (− 6.42, − 3.36)	< 0.0001	0.2101	0.9515
Glutamine*	18.64 (16.04, 21.67)	8.82 (7.39, 10.53)	21.41 (18.46, 24.83)	10.13 (8.61, 11.92)	< 0.0001	0.1356	0.4880
Glycine	8.97 (7.36, 10.58)	4.14 (2.28, 5.99)	10.63 (9.04, 12.22)	5.79 (4.05, 7.53)	< 0.0001	0.0945	0.4103
Histidine	2.38 (2.04, 2.72)	1.58 (1.19, 1.98)	2.61 (2.28, 2.95)	1.82 (1.45, 2.19)	0.0003	0.2569	0.9511
Isoleucine*	7.25 (6.23, 8.43)	3.02 (2.53, 3.59)	8.48 (7.3, 9.84)	3.53 (3.00, 4.16)	< 0.0001	0.0923	0.8945
Leucine	12.17 (11, 13.34)	5.59 (4.24, 6.94)	12.77 (11.61, 13.92)	6.19 (4.92, 7.46)	< 0.0001	0.4053	0.8465
Lysine	6.88 (6.07, 7.69)	3.72 (2.78, 4.65)	7.79 (6.99, 8.59)	4.62 (3.75, 5.50)	< 0.0001	0.0689	0.8776
Methionine*	2.32 (1.88, 2.86)	1.12 (0.88, 1.43)	3.16 (2.57, 3.88)	1.53 (1.22, 1.91)	< 0.0001	0.0180	0.9275
Ornithine	0.97 (0.57, 1.37)	0.54 (0.08, 1.00)	1.22 (0.83, 1.62)	0.80 (0.37, 1.23)	0.0846	0.2900	0.9471
Phenylalanine	4.55 (3.89, 5.21)	1.92 (1.16, 2.68)	5.61 (4.96, 6.26)	2.98 (2.27, 3.69)	< 0.0001	0.0098	0.2213
Proline*	6.15 (5.24, 7.22)	2.68 (2.23, 3.23)	7.92 (6.76, 9.28)	3.46 (2.91, 4.11)	< 0.0001	0.0111	0.3452
Serine	− 2.85 (− 4.72, − 0.98)	− 0.64 (− 2.80, 1.52)	− 2.67 (− 4.52, − 0.83)	− 0.46 (− 2.49, 1.56)	0.0589	0.8765	0.9173
Taurine	0.69 (0.01, 1.37)	0.49 (− 0.29, 1.27)	0.02 (− 0.65, 0.69)	− 0.19 (− 0.92, 0.55)	0.6259	0.1050	0.6935
Threonine	7.06 (5.9, 8.23)	3.61 (2.27, 4.95)	7.76 (6.61, 8.90)	4.30 (3.05, 5.56)	< 0.0001	0.3286	0.9378
Tryptophan*	1.28 (1.01, 1.61)	0.55 (0.42, 0.72)	1.44 (1.15, 1.81)	0.62 (0.48, 0.80)	< 0.0001	0.3891	0.3750
Tyrosine	4.53 (3.86, 5.2)	1.85 (1.08, 2.63)	5.27 (4.61, 5.93)	2.59 (1.87, 3.32)	< 0.0001	0.0739	0.1772
Valine	12.03 (10.32, 13.74)	6.37 (4.40, 8.34)	13.22 (11.53, 14.90)	7.56 (5.71, 9.40)	< 0.0001	0.2556	0.8525
Umbilical oxygen and amino acid uptake rates relative to fetal weight (μmol/min/kg)
Oxygen*	351.61 (333.36, 370.86)	301.13 (283.53, 319.83)	363.09 (344.76, 382.4)	310.97 (293.86, 329.07)	< 0.0001	0.3188	0.6840
Alanine	2.86 (2.46, 3.27)	2.21 (1.75, 2.68)	3.29 (2.89, 3.69)	2.64 (2.20, 3.07)	0.0104	0.0846	0.1548
Arginine	2.50 (2.22, 2.77)	1.69 (1.37, 2.01)	2.80 (2.53, 3.07)	1.99 (1.69, 2.29)	< 0.0001	0.0748	0.5150
Asparagine	1.09 (0.78, 1.4)	1.33 (0.98, 1.69)	0.79 (0.48, 1.09)	1.03 (0.70, 1.36)	0.2033	0.1125	0.3287
Aspartate	− 0.06 (− 0.17, 0.06)	− 0.06 (− 0.20, 0.07)	− 0.02 (− 0.13, 0.09)	− 0.03 (− 0.15, 0.10)	0.9205	0.5961	0.8031
Citrulline	− 0.06 (− 0.35, 0.22)	0.30 (− 0.03, 0.63)	− 0.06 (− 0.34, 0.23)	0.31 (0.00, 0.62)	0.0412	0.9647	0.1078
Cystine	0.07 (0.00, 0.14)	0.09 (0.01, 0.17)	0.13 (0.06, 0.20)	0.15 (0.07, 0.23)	0.6864	0.1593	0.9808
Glutamate	− 3.15 (− 3.56, − 2.73)	− 1.78 (− 2.26, − 1.30)	− 3.51 (− 3.93, − 3.10)	− 2.15 (− 2.60, − 1.70)	< 0.0001	0.1502	0.3734
Glutamine*	5.79 (5.15, 6.52)	4.65 (4.05, 5.35)	6.40 (5.70, 7.19)	5.14 (4.52, 5.84)	0.0036	0.1710	0.3270
Glycine*	2.36 (1.98, 2.81)	1.86 (1.52, 2.28)	2.85 (2.4, 3.38)	2.25 (1.86, 2.71)	0.0302	0.0811	0.8226
Histidine	0.70 (0.60, 0.80)	0.77 (0.65, 0.88)	0.79 (0.69, 0.89)	0.85 (0.74, 0.96)	0.3032	0.1761	0.2656
Isoleucine*	2.24 (1.98, 2.53)	1.56 (1.35, 1.79)	2.55 (2.26, 2.88)	1.77 (1.56, 2.02)	< 0.0001	0.0807	0.8945
Leucine	3.69 (3.37, 4.02)	2.81 (2.44, 3.18)	3.79 (3.47, 4.11)	2.91 (2.56, 3.26)	< 0.0001	0.6161	0.5456
Lysine	2.06 (1.85, 2.28)	1.89 (1.65, 2.14)	2.33 (2.12, 2.55)	2.16 (1.93, 2.40)	0.2057	0.0421	0.3436
Methionine*	0.72 (0.59, 0.87)	0.58 (0.46, 0.72)	0.95 (0.79, 1.15)	0.77 (0.62, 0.95)	0.0767	0.0179	0.7731
Ornithine	0.27 (0.13, 0.41)	0.27 (0.11, 0.43)	0.39 (0.25, 0.53)	0.39 (0.23, 0.54)	0.9918	0.1791	0.4196
Phenylalanine	1.36 (1.18, 1.54)	1.02 (0.81, 1.23)	1.66 (1.48, 1.84)	1.33 (1.13, 1.52)	0.0034	0.0068	0.6058
Proline*	1.90 (1.63, 2.2)	1.38 (1.16, 1.65)	2.38 (2.06, 2.76)	1.74 (1.48, 2.05)	0.001	0.0143	0.4438
Serine	− 0.92 (− 1.49, − 0.36)	− 0.29 (− 0.94, 0.36)	− 0.80 (− 1.35, − 0.24)	− 0.16 (− 0.77, 0.45)	0.0715	0.7093	0.9961
Taurine	0.23 (0.02, 0.45)	0.19 (− 0.06, 0.43)	0.00 (− 0.21, 0.20)	− 0.05 (− 0.28, 0.18)	0.7106	0.0672	0.4601
Threonine*	1.76 (1.44, 2.16)	1.56 (1.23, 1.97)	2.07 (1.69, 2.53)	1.83 (1.47, 2.28)	0.3294	0.1982	0.6802
Tryptophan*	0.39 (0.32, 0.48)	0.28 (0.23, 0.36)	0.43 (0.36, 0.53)	0.31 (0.25, 0.38)	0.0073	0.4331	0.3867
Tyrosine*	1.27 (1.09, 1.49)	0.89 (0.74, 1.07)	1.41 (1.21, 1.65)	0.99 (0.83, 1.17)	0.0005	0.2973	0.7439
Valine	3.60 (3.07, 4.13)	3.17 (2.56, 3.78)	3.97 (3.45, 4.49)	3.54 (2.96, 4.11)	0.1856	0.2557	0.5726
Uterine oxygen and amino acid uptake rates (μmol/min)
Oxygen	1925.38 (1697.02, 2153.74)	993.88 (616.59, 1371.17)	2055.05 (1771.62, 2338.49)	1123.55 (811.70, 1435.40)	< 0.0001	0.4369	0.3792
Alanine	5.66 (3.60, 7.72)	3.59 (0.19, 7.00)	5.64 (3.09, 8.20)	3.58 (0.77, 6.39)	0.214	0.9921	0.3516
Arginine	12.04 (8.52, 15.57)	8.99 (3.17, 14.82)	9.47 (5.09, 13.84)	6.42 (1.60, 11.23)	0.2818	0.3182	0.1418
Asparagine	1.97 (0.53, 3.40)	1.62 (− 0.75, 3.99)	2.36 (0.58, 4.14)	2.01 (0.05, 3.97)	0.7619	0.7060	0.9341
Aspartate	− 0.91 (− 1.48, − 0.33)	− 0.40 (− 1.35, 0.55)	− 0.73 (− 1.45, − 0.02)	− 0.23 (− 1.01, 0.56)	0.2727	0.6801	0.9564
Citrulline	5.66 (2.34, 8.98)	4.34 (− 1.14, 9.82)	5.99 (1.87, 10.10)	4.67 (0.14, 9.20)	0.6184	0.8928	0.5253
Cystine	0.00 (− 0.76, 0.75)	0.76 (− 0.48, 2.01)	0.07 (− 0.87, 1.00)	0.83 (− 0.20, 1.86)	0.2086	0.8982	0.4157
Glutamate	− 2.25 (− 4.60, 0.11)	− 2.09 (− 5.98, 1.81)	− 2.19 (− 5.12, 0.73)	− 2.03 (− 5.25, 1.19)	0.9312	0.9745	0.9284
Glutamine	12.30 (8.38, 16.23)	10.48 (3.99, 16.96)	13.74 (8.87, 18.61)	11.91 (6.55, 17.28)	0.5599	0.6155	0.1830
Glycine	− 5.36 (− 10.77, 0.05)	0.37 (− 8.57, 9.31)	− 7.26 (− 13.98, − 0.55)	− 1.53 (− 8.92, 5.86)	0.1897	0.6289	0.1837
Histidine	2.65 (1.82, 3.49)	1.87 (0.48, 3.25)	1.92 (0.88, 2.96)	1.13 (− 0.01, 2.27)	0.2426	0.2322	0.8716
Isoleucine*	9.61 (7.16, 12.89)	5.25 (3.23, 8.54)	11.76 (8.16, 16.93)	6.43 (4.30, 9.61)	0.0142	0.3481	0.1169
Leucine*	11.97 (9.16, 15.65)	6.41 (4.12, 9.97)	14.49 (10.39, 20.20)	7.75 (5.38, 11.18)	0.0059	0.3307	0.5231
Lysine	6.29 (4.58, 8.01)	5.35 (2.52, 8.19)	5.95 (3.82, 8.08)	5.02 (2.67, 7.36)	0.494	0.7871	0.5576
Methionine	3.35 (2.17, 4.53)	2.21 (0.26, 4.16)	2.28 (0.81, 3.74)	1.14 (− 0.47, 2.75)	0.2311	0.2193	0.3284
Ornithine	6.07 (4.28, 7.86)	4.62 (1.65, 7.58)	4.59 (2.36, 6.81)	3.14 (0.69, 5.58)	0.3136	0.2602	0.6135
Phenylalanine	2.50 (1.36, 3.63)	2.07 (0.20, 3.95)	3.47 (2.06, 4.88)	3.04 (1.49, 4.60)	0.6377	0.2443	0.6835
Proline*	9.66 (7.04, 13.25)	4.53 (2.68, 7.63)	8.67 (5.86, 12.84)	4.06 (2.64, 6.26)	0.0049	0.6408	0.6434
Serine	7.92 (5.37, 10.46)	4.23 (0.02, 8.44)	8.51 (5.35, 11.67)	4.82 (1.35, 8.30)	0.0764	0.7491	0.9997
Taurine	− 0.42 (− 2.51, 1.67)	0.43 (− 3.02, 3.89)	− 3.11 (− 5.7, − 0.52)	− 2.26 (− 5.11, 0.59)	0.6091	0.0834	0.2527
Threonine	8.27 (5.88, 10.67)	3.61 (− 0.34, 7.56)	7.92 (4.95, 10.89)	3.26 (− 0.01, 6.52)	0.0192	0.8398	0.9553
Tryptophan	2.53 (1.69, 3.37)	2.1 (0.72, 3.49)	2.80 (1.76, 3.84)	2.37 (1.22, 3.52)	0.5249	0.6630	0.9963
Tyrosine	3.81 (2.22, 5.41)	4.74 (2.10, 7.38)	3.72 (1.74, 5.70)	4.65 (2.47, 6.83)	0.4675	0.9353	0.6302
Valine	16.28 (12.40, 20.16)	7.46 (1.05, 13.87)	18.96 (14.14, 23.78)	10.14 (4.84, 15.44)	0.0072	0.3452	0.7676
Uterine oxygen and amino acid uptake rates relative to placental weight (μmol/min/kg)
Oxygen*	6.15 (5.57, 6.79)	5.43 (4.58, 6.44)	5.89 (5.2, 6.67)	5.20 (4.46, 6.07)	0.1458	0.5524	0.5414
Alanine	17.17 (10.99, 23.36)	17.58 (7.20, 27.96)	16.79 (9.10, 24.48)	17.20 (8.26, 26.14)	0.9352	0.9323	0.1599
Arginine	43.62 (28.13, 59.11)	22.21 (− 12.43, 56.85)	22.38 (2.39, 42.38)	52.73 (28.24, 77.22)	#	#	0.0406
Asparagine	5.94 (0.73, 11.15)	9.69 (0.95, 18.43)	6.74 (0.26, 13.21)	10.49 (2.96, 18.02)	0.3804	0.8342	0.9321
Aspartate	− 2.80 (− 4.76, − 0.84)	− 2.15 (− 5.44, 1.14)	− 1.87 (− 4.30, 0.57)	− 1.22 (− 4.05, 1.61)	0.6868	0.5157	0.7577
Citrulline	19.82 (2.49, 37.14)	16.51 (− 12.58, 45.59)	15.53 (− 6.01, 37.07)	12.22 (− 12.83, 37.27)	0.8149	0.7348	0.9343
Cystine	0.26 (− 2.24, 2.76)	3.69 (− 0.50, 7.88)	− 0.07 (− 3.17, 3.04)	3.36 (− 0.25, 6.97)	0.0999	0.8567	0.3941
Glutamate	− 6.79 (− 14.87, 1.28)	− 10.89 (− 24.45, 2.67)	− 7.94 (− 17.98, 2.10)	− 12.05 (− 23.72, − 0.37)	0.5348	0.8449	0.5597
Glutamine	40.20 (27.32, 53.09)	57.20 (35.56, 78.83)	40.33 (24.31, 56.35)	57.32 (38.69, 75.95)	0.1134	0.9895	0.2054
Glycine	− 16.64 (− 36.50, 3.21)	− 6.59 (− 39.92, 26.75)	− 22.13 (− 46.81, 2.56)	− 12.07 (− 40.78, 16.64)	0.5358	0.7052	0.4457
Histidine	8.53 (5.62, 11.44)	7.04 (2.15, 11.93)	5.80 (2.18, 9.42)	4.31 (0.10, 8.52)	0.5317	0.2056	0.9194
Isoleucine*	34.72 (27.08, 44.53)	18.89 (10.84, 32.95)	29.77 (21.6, 41.04)	47.91 (32.33, 70.99)	#	#	0.0091
Leucine	43.2 (34.54, 51.86)	41.91 (27.37, 56.45)	46.01 (35.24, 56.78)	44.72 (32.20, 57.25)	0.8552	0.6565	0.0886
Lysine	20.64 (15.18, 26.10)	26.92 (17.75, 36.09)	18.05 (11.26, 24.84)	24.33 (16.43, 32.23)	0.1658	0.5174	0.3043
Methionine	10.67 (7.03, 14.3)	9.03 (2.92, 15.13)	6.45 (1.93, 10.97)	4.81 (− 0.45, 10.07)	0.5817	0.1193	0.2621
Ornithine	19.27 (12.62, 25.92)	26.98 (15.82, 38.14)	14.19 (5.93, 22.46)	21.91 (12.29, 31.52)	0.1617	0.2995	0.1621
Phenylalanine	8.07 (3.96, 12.19)	6.84 (− 2.36, 16.05)	8.90 (3.58, 14.21)	21.2 (14.69, 27.7)	#	#	0.0437
Proline*	31.33 (23.51, 41.74)	27.98 (17.28, 45.29)	26.31 (18.41, 37.59)	23.49 (15.51, 35.58)	0.6293	0.4068	0.2289
Serine	25.05 (16.9, 33.2)	24.73 (11.05, 38.41)	25.06 (14.93, 35.19)	24.74 (12.95, 36.52)	0.9607	0.9986	0.5297
Taurine	− 1.56 (− 8.17, 5.05)	− 0.33 (− 11.43, 10.77)	− 6.66 (− 14.88, 1.56)	− 5.43 (− 14.99, 4.14)	0.8189	0.2948	0.1071
Threonine	26.91 (18.78, 35.04)	21.26 (7.61, 34.91)	23.21 (13.11, 33.32)	17.57 (5.81, 29.32)	0.3972	0.5345	0.7375
Tryptophan	8.00 (4.89, 11.10)	13.23 (8.01, 18.44)	8.01 (4.15, 11.87)	13.23 (8.75, 17.72)	0.0459	0.9966	0.7736
Tyrosine	11.20 (6.93, 15.47)	21.13 (13.97, 28.3)	11.64 (6.33, 16.95)	21.57 (15.40, 27.75)	0.0073	0.8875	0.1498
Valine	53.77 (40.60, 66.93)	42.27 (20.17, 64.37)	55.08 (38.71, 71.44)	43.58 (24.54, 62.61)	0.2886	0.8916	0.8959

The effect of sex on the association between parameters and experimental placental insufficiency–induced IUGR was tested using linear models with an interaction term

^*The data were log transformed prior to analysis

^#A significant interaction effect (P < 0.05) with the following P values for individual means comparisons: arginine: IUGR females vs. control females (P = 0.2600), IUGR males vs. control males (P = 0.0600), control females vs. control males (P = 0.1000), and IUGR females vs. IUGR males (P = 0.1500); isoleucine: IUGR females vs. control females (P = 0.0500), IUGR males vs. control males (P = 0.0700), control females vs. control males (P = 0.4400), and IUGR females vs. IUGR males (P = 0.0100); phenylalanine: IUGR females vs. control females (P = 0.8000), IUGR males vs. control males (P = 0.0100), control females vs. control males (P = 0.8000), and IUGR females vs. IUGR males (P = 0.0100)

Results

Metabolic studies were performed at the gestational age of 133 ± 0 days. This was the same for control animals, IUGR animals, animals with male fetuses, and animals with female fetuses. The main goal of this study was to determine if the experimental procedure to produce IUGR impacted pregnancies with male fetuses differently than pregnancies with female fetuses. Of all parameters analyzed, the interaction between IUGR and fetal sex was significant for three: uterine uptake rates normalized to placental weight of isoleucine, phenylalanine, and arginine (Table 1). There were also very few parameters that demonstrated a difference between male and female fetuses when adjusting for the effect of placental insufficiency (Table 1). Differences between male and female fetuses in umbilical amino acid uptake rates were identified for proline, methionine, and phenylalanine whether normalized to fetal weight or not. Additionally, absolute (not normalized to fetal weight) umbilical arginine uptake rate and umbilical lysine uptake rate relative to fetal weight were higher in male fetuses compared to female fetuses. We also found that male fetuses were not heavier than female fetuses in our cohort (Table 1). Even a post hoc analysis of just control animals did not demonstrate a difference between female and male fetuses. Female control fetuses weighed 5.3% less than male control fetuses (P = 0.1262; males 3363 g, 95% confidence interval: 3183–3543 g; females 3185, 95% confidence interval: 3038–3332 g). Fetal arterial plasma cortisol and norepinephrine were both higher in female fetuses compared to male fetuses. As expected, we identified numerous parameters that were statistically different between the control and IUGR groups (Table 1).

We next correlated umbilical uptake rates of the various amino acids with fetal weight. The relationships of individual amino acid umbilical uptake rates to fetal weight are shown in Table 2, ranked by their Pearson’s correlation coefficient (R), highest to lowest. Umbilical uptake of the essential branched-chain amino acid (BCAA) leucine had the highest correlation with fetal weight (Fig. 1A). The association between umbilical leucine uptake and fetal weight was such that for every kilogram increase in fetal weight, umbilical leucine uptake increased by 4.80 μmol/min (95% confidence interval: 4.12–5.48). Umbilical uptake rates of the other BCAAs, isoleucine and valine, were also highly correlated with fetal weight. Umbilical uptake of the conditionally essential amino acid glutamine had the largest estimated association such that for every kilogram increase in fetal weight, umbilical glutamine uptake increased by 8.31 μmol/min (95% confidence interval: 6.80–9.82, Fig. 1B). Umbilical uptake of all essential and five of the seven conditionally essential amino acids had highly significant correlations with fetal weight (P < 1 × 10⁻⁹). Of all the non-essential amino acids, only the correlation of the umbilical uptake of alanine with fetal weight was highly significant (P = 5.60 × 10⁻¹⁴) with an estimated association such that for every kilogram increase in fetal weight, umbilical alanine uptake increased by 4.30 μmol/min (95% confidence interval: 3.34–5.26, Fig. 1C). Umbilical uptake of the non-essential amino acid glutamate was the only uptake rate that correlated negatively with fetal weight with a statistically significant relationship (r = − 0.763, P = 1.40 × 10⁻¹⁸; Fig. 1D). A negative umbilical uptake rate indicates that the placenta is taking up glutamate from the fetal circulation. The estimated association is such that for every kilogram increase in fetal weight, placental glutamate uptake from the fetal circulation increased by 4.92 μmol/min (95% confidence interval: 4.04–5.80). This means that smaller fetuses had lower placental uptake of glutamate from the fetal circulation.

Table 2.

Linear regression characteristics of umbilical amino acid uptake rates and fetal weights

Amino acid		Pearson’s correlation (R)	R 2	P value	Slope	95% confidence interval
Leucine	Essential	0.829	0.687	3.43 × 10−24	4.80	4.12–5.48
Arginine	Conditionally essential	0.798	0.636	2.99 × 10−21	3.68	3.09–4.26
Glutamine	Conditionally essential	0.759	0.576	4.26 × 10−18	8.31	6.80–9.82
Phenylalanine	Essential	0.754	0.569	6.09 × 10−18	2.16	1.77–2.56
Lysine	Essential	0.748	0.559	1.62 × 10−17	2.61	2.12–3.10
Isoleucine	Essential	0.745	0.556	2.34 × 10−17	3.46	2.81–4.12
Tyrosine	Conditionally essential	0.713	0.509	2.19 × 10−15	2.10	1.67–2.54
Proline	Conditionally essential	0.689	0.474	4.49 × 10−14	3.03	2.36–3.71
Alanine	Non-essential	0.687	0.472	5.60 × 10−14	4.30	3.34–5.26
Valine	Essential	0.676	0.457	1.93 × 10−13	4.77	3.68–5.86
Glycine	Conditionally essential	0.657	0.432	1.45 × 10−12	4.46	3.39–5.54
Histidine	Essential	0.648	0.420	3.91 × 10−12	0.88	0.66–1.10
Tryptophan	Essential	0.635	0.403	1.40 × 10−11	0.74	0.55–0.93
Methionine	Essential	0.597	0.356	4.35 × 10−10	1.31	0.94–1.69
Threonine	Essential	0.594	0.353	5.27 × 10−10	2.72	1.94–3.49
Cystine	Conditionally essential	0.294	0.086	0.0047	0.25	0.08–0.42
Ornithine	Non-essential	0.274	0.075	0.0085	0.39	0.10–0.68
Asparagine	Non-essential	0.216	0.047	0.0394	0.78	0.04–1.53
Taurine	Conditionally essential	0.026	0.001	0.8093	0.06	− 0.44 to 0.56
Citrulline	Non-essential	− 0.077	0.006	0.4708	− 0.23	− 0.87 to 0.40
Aspartate	Non-essential	− 0.079	0.006	0.4566	− 0.11	− 0.41 to 0.18
Serine	Non-essential	− 0.170	0.029	0.1074	− 1.12	− 2.49 to 0.25
Glutamate	Non-essential	− 0.763	0.583	1.40 × 10−18	− 4.92	− 5.80 to − 4.04

Pearson’s correlation was used to assess the correlation between umbilical amino acid uptake rates and fetal weight

Fig. 1.

Correlation of umbilical amino acid uptake rates and fetal weights. Individual fetal weights are plotted as a function of leucine (A), glutamine (B), alanine (C), and glutamate (D) umbilical uptake rates for control (females = open circles, males = open squares, sex not recorded = open triangles) and IUGR (females = closed circles, males = closed squares, sex not recorded = closed triangles) fetuses. Pearson’s correlation (R), R², the slope of the relationship with 95% confidence intervals, and the P value of the association are provided below each graph

We next correlated fetal arterial plasma concentrations of the five hormones that were measured to fetal weight (Table 3, Fig. 2). When considering all animals, two hormone concentrations had a positive correlation with fetal weight, insulin, and IGF-1. Of these two, IGF-1 was the most highly correlated. The association between fetal arterial plasma IGF-1 concentrations and fetal weight was such that for every kilogram increase in fetal weight, IGF-1 concentrations increased by 36.54 ng/mL (95% confidence interval: 30.01–43.03). Three hormone concentrations had a negative correlation with fetal weight: norepinephrine, cortisol, and glucagon. Of these, norepinephrine was the most highly negatively correlated. The association between fetal arterial plasma norepinephrine concentrations and fetal weight was such that for every kilogram decrease in fetal weight, norepinephrine concentrations increased by 701.82 pg/mL (95% confidence interval: 523.98–879.66). We also tested whether the association between fetal weight and each hormone was different for control and IUGR pregnancies (Table 3).

Table 3.

Linear regression characteristics of fetal arterial hormones and fetal weights

Hormone	Pearson’s correlation (R)	R 2	P value	Slope	95% confidence interval
Insulin-like growth factor 1*
All animals	0.776	0.602	2.65 × 10−18	36.54	30.01–43.03
Control	0.360	0.130	8.81 × 10−3	22.98	6.05–39.91
IUGR	0.828	0.686	2.69 × 10−9	42.16	31.71–52.60
Insulin*
All animals	0.421	0.177	1.43 × 10−5	0.13	0.07–0.18
Control	− 0.127	0.016	0.3466	− 0.08	− 2.30 to 0.09
IUGR	0.410	0.168	7.80 × 10−3	0.06	0.02–0.10
Norepinephrine*
All animals	− 0.629	0.395	7.06 × 10−12	− 701.82	− 879.66 to − 523.98
Control	− 0.239	0.057	0.0703	− 212.78	− 443.75 to 18.20
IUGR	− 0.505	0.255	1.21 × 10−3	− 894.85	− 1411.16 to − 378.54
Cortisol
All animals	− 0.408	0.166	3.37 × 10−5	− 11.28	− 16.42 to − 6.14
Control	− 0.107	0.011	0.4228	− 3.72	− 12.96 to 5.51
IUGR	− 0.355	0.126	2.65 × 10−2	− 15.85	− 29.74 to − 1.96
Glucagon
All animals	− 0.373	0.139	4.31 × 10−3	− 19.62	− 32.83 to − 6.41
Control	− 0.136	0.018	0.4104	− 10.60	− 36.39 to 15.19
IUGR	− 0.366	0.025	0.1361	− 24.58	− 57.78 to 8.62

Pearson’s correlation was used to assess the correlation between fetal arterial hormone concentrations and fetal weight for all animals combined, just control animals, and just IUGR animals

^*A significant differences between the regression lines for control vs. IUGR animals (P = 0.0006, IGF-1; P = 0.0010, insulin; P = 0.0242, norepinephrine; P = 0.3279, cortisol; P = 0.1531, glucagon)

Fig. 2.

Correlation of fetal arterial plasma hormone concentrations and fetal weights. Individual fetal weights are plotted as a function of the following fetal arterial plasma concentrations: IGF-1 (A), insulin (B), norepinephrine (C), cortisol (D), and glucagon (E) for control (females = open circles, males = open squares, sex not recorded = open triangles) and IUGR (females = closed circles, males = closed squares, sex not recorded = closed triangles) fetuses. Pearson’s correlation (R), R², the slope of the relationship with 95% confidence intervals, and the P value of the association are provided in Table 3. The regression line for all animals is shown as a solid bold line. The regression line for just control animals is shown as a thinner solid line, and the regression line for just IUGR animals is shown as a dashed line

Discussion

Pregnant sheep have been used to understand normal placental function and fetal physiology as well as to model various complications of human pregnancy [12]. Our research group has extensively studied the regulation of fetal growth in the latter half of pregnancy, including the role of placental amino acid supply in this regulation. As part of these studies, we have utilized a model of placental insufficiency in which pregnant sheep are exposed to elevated ambient temperatures during the middle part of gestation. This exposure results in a small and poorly functioning placenta and fetal IUGR [30]. While we have previously demonstrated lower umbilical uptake rates for several different amino acids in this model, we have never uncovered an impact of fetal sex on umbilical amino acid uptake [16–19]. By combining data from previously published and ongoing studies, we have determined that fetal sex does not impact the effect of IUGR for any umbilical amino acid uptake rates. In fact, this was true for all but three outcomes: uterine uptake rates normalized to placental weight of isoleucine, phenylalanine, and arginine. Furthermore, the only other amino acid uptake rates that demonstrated a difference between male and female fetuses were the umbilical uptake rates of proline, methionine, phenylalanine, lysine, and arginine, and there was no interaction between fetal sex and IUGR for these rates.

This is the largest single analysis conducted on this topic and provides strong evidence for a lack of sex differences in the outcomes we have reported in the current manuscript. These outcomes include not only umbilical and uterine amino acid uptake rates, but also umbilical and uterine blood flows, oxygen uptake rates, placental and fetal weights, and fetal arterial plasma insulin, IGF-1, and glucagon concentrations. Fetal arterial plasma concentrations of cortisol and norepinephrine demonstrated differences based on fetal sex with the concentrations of both of these stress response hormones higher in females than in males, regardless of whether the fetuses were IUGR or not. Male newborn intensive care unit patients have worse outcomes than female patients, including IUGR and preterm patients [31–33]. If human female fetuses have higher cortisol and norepinephrine concentrations than male fetuses in response to similar prenatal insults, these higher concentrations may better prepare the female newborns for postnatal adaptation. Especially relevant would be the maturation of fetal lung development by cortisol [34] and the slowing of fetal growth by norepinephrine in order to decrease progression of fetal hypoxemia during placental insufficiency [35]. Our results with normal fetal sheep and fetal sheep subjected to chronic hypoxemia and placental insufficiency stand in contrast to other studies demonstrating that the acute fetal cortisol response to hypoxia is higher in male fetal sheep compared to female fetal sheep [36].

In our study, we did not find that fetal sex modified the impact of IUGR on fetal weight. Moreover, we did not find any statistical differences between the weights of male and female fetuses. This was true even when restricting our analysis to just the control fetuses. Mean weights of female control fetuses were 5.3% less than male control fetuses, failing to reach statistical significance. When considering the variability of all control fetuses, we would need close to 93 control male and 93 control female fetuses in order to detect a statistically significant difference (P < 0.05) in a fetal weight of 5.3% between these two groups with a power of 80%. In newborn lambs of various breeds, females weigh between 5 and 10% less than males [25, 26]. This typically requires large sample sizes to reach significance, as the effect size of biological sex is about the same as the standard deviation of normal lamb birth weights. For example, in a study of Southdown and Romney purebred and crossed lambs, the difference between male and female birth weights and standard deviation of birth weights were reported as 460 g and 490 g, respectively [26]. While direct numerical comparisons of fetal weights in the breed of sheep used in our studies with other breeds of sheep are not possible, by looking at the effect size as a percent of male weight, it is clear that the magnitude of the difference between males and females may become larger as the final 10% of gestation progresses. However, given the similarities between effect size and the standard deviation of both the fetal lambs in our studies and newborn lambs, the numbers of animals to detect a statistically significant difference in male and female weights at any point in gestation are extremely high. Thus, while it is true that our study of animals at gestational day 133 may have been too early to detect any subtle but true differences in fetal weights especially considering that our studies were performed before the typical rise in prenatal cortisol concentrations in sheep [37], it is also evident that even at the time of spontaneous birth, true differences in newborn lamb weight based on fetal sex may be difficult to discern in most studies of fetal and newborn physiology and development given the numbers of animals typically utilized in these studies. These observations in fetal and newborn lambs mirror the observations in human newborns. In studies with enough subjects, a statistically significant difference between male and female weights can be identified, with males weighing more than females [23, 24]. However, the difference between male and female birth weights is even smaller in humans than in sheep. For example, in a study of 9413 newborns, females were found to weigh 121 g less than males. This represents a difference of 3.5% in female birth weights relative to male birth weights and compares to a standard deviation of the mean for birth weights in the entire cohort of 512 g [24].

Unlike the situation for differences based on fetal sex, we identified widespread differences based on the presence or absence of experimental IUGR. In many cases, these differences demonstrated a large effect size and highly statistically significant relationships. These results confirm relationships we have previously identified between control and IUGR pregnancies following exposure of the mother to elevated ambient temperatures during mid-gestation. These include differences in placental and fetal weight, uterine and umbilical blood and plasma flows, and fetal arterial blood oxygen and plasma IGF-1, insulin, norepinephrine, and cortisol concentrations [30, 38]. Additionally, we have previously demonstrated lower umbilical amino acid uptake rates in the IUGR pregnancies [17–19, 39]. Differences between control and IUGR pregnancies are also consistent with human pregnancies complicated placental insufficiency and IUGR [12, 30, 40].

Some previous studies have demonstrated statistically significantly lower umbilical uptake rates of oxygen normalized to fetal weight, and others have not in this particular model of IUGR [39, 41, 42]. This is an important rate to consider because it is equal to the fetal oxygen utilization rate and is thus a measure of fetal energy expenditure. In our study a 14% lower umbilical oxygen uptake rate was readily apparent in the IUGR fetuses (P < 0.0001).

We also demonstrated with the current analysis about 50% higher fetal arterial plasma glucagon concentrations in the IUGR fetuses relative to control fetuses (P = 0.0331). While higher mean glucagon concentrations have been reported for this model of IUGR before, the results have not always been statistically significant [38, 43]. Even in the current analysis, the variability is high relative to the effect size, indicating that prior studies were likely underpowered to uncover this relationship when defining significance as P < 0.05. While studies that have measured glucagon concentrations in human fetuses are limited compared to other hormones such as IGF-1 and insulin, there are reports of elevated glucagon concentrations in human IUGR fetuses, especially those with abnormal Doppler ultrasound findings indicating a more severe case of placental insufficiency [44].

Interestingly, umbilical amino acid uptake rates represent a continuum across the spectrum of weights in both control and IUGR pregnancies. The results are not bimodal. This is most easily seen in the graphs presented in Fig. 1. The most highly correlated umbilical amino acid uptake rate with fetal weight was for leucine. Leucine is an essential branched-chain amino acid. Leucine transport into the fetal circulation is impaired in human pregnancies complicated by placental insufficiency and IUGR. In fact, one study in human pregnant women demonstrated that the degree to which placental leucine transport into the fetal circulation is impaired correlates with the severity of placental insufficiency and IUGR [4]. This finding is largely duplicated by our current analysis, thus supporting a primary role for placental leucine transport in maintaining normal weight pregnancies. The rest of the amino acids with uptake rates that were highly correlated to fetal weight were either essential or conditionally essential amino acids for the fetus [45, 46]. Alanine was the only non-essential amino acid with an uptake rate that was highly correlated to fetal weight. Alanine may be particularly important for small and growth-restricted fetuses. It has been proposed that when glucose transfer to the fetus is limited by placental insufficiency, the fetus may adapt by preferentially decreasing alanine utilization in tissues like the skeletal muscle so that alanine can be spared for utilization by the fetal liver in the process of gluconeogenesis [47]. In fact, in the smallest growth-restricted fetuses, the hindlimb releases alanine into the fetal circulation and circulating plasma alanine concentrations are higher [48].

Another noteworthy pair of umbilical amino acid uptake rates to consider is those for glutamine and glutamate. In the normal fetus, the placenta removes glutamate from the fetal circulation. Thus, negative uptake rates are demonstrated in the current analysis. This is part of a glutamate-glutamine exchange between the fetal liver and placenta [49]. In the case of severe IUGR, the fetus diverts the carbons required to synthesize glutamate into glucose through the process of gluconeogenesis [47]. Without glutamate produced by the fetal liver, the placenta no longer clears as much glutamate from the fetal circulation to convert into glutamine [49]. Thus, in the smallest IUGR fetuses, the umbilical glutamine uptake rate becomes very small. And like the situation for alanine, glutamine utilization by the fetal muscles becomes restricted and the smallest fetuses release glutamine from their muscle into the fetal circulation, as they do for alanine [48].

We also plotted various fetal arterial plasma hormone concentrations by fetal weight. IGF-1 and insulin are two of the more dominant anabolic fetal growth hormones [50, 51]. The regulation of these hormones starts with placental nutrient supply stimulating pancreatic β-cell secretion of insulin, which then, in turn, stimulates the liver to secrete IGF-1. IGF-1 concentrations are also under direct nutrient regulation [52, 53]. In the current analysis, IGF-1 is more highly correlated with fetal weight than insulin. This is likely due to the fact that fetal sheep insulin concentrations are more responsive to acute changes in nutrient supply and fetal arterial nutrient concentrations than fetal IGF-1 concentration [53, 54]. Thus, fetal arterial insulin concentrations likely reflect the immediate nutrient supply of the fetus whereas IGF-1 concentrations may reflect the more chronic supply of nutrients. Of the three growth-suppressing hormones we measured, norepinephrine was the most negatively correlated with fetal weight, again highlighting its important role in modulating concentrations of the anabolic growth factor, insulin, during times of relatively low oxygen concentrations [35].

Conclusion

The complexity of fetal and placental physiology and the pathogenesis of the developmental origins of adult diseases mandate consideration of fetal sex. Many of the long-term outcomes being studied in the adults following fetal and developmental insults show a preference for one sex over the other. To the extent that the placenta is a major determinant of these long-term outcomes, the role of fetal sex in dictating placental function and those long-term outcomes should be addressed. Our analysis demonstrates a general lack of differences between male and female fetuses for multiple parameters important for the regulation of fetal growth at 133 dga. These include placental weight, uterine and umbilical blood flow, uterine and umbilical oxygen and amino acid uptake rates, and fetal arterial plasma IGF-1 and insulin concentrations. This is consistent with the lack of differences in fetal weight based on fetal sex. The higher fetal arterial norepinephrine and cortisol concentrations in females relative to males may have important implications for linking fetal growth patterns with long-term metabolic outcomes. Although we cannot rule out the possibility that larger numbers of animals would have detected statistically significant lower fetal weight in female fetuses and other outcome differences based on fetal sex, it seems the inherent variability in these measurements is a limitation for detecting differences based on fetal sex.

Data Availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.