Test of long-term uterine survival after allogeneic transplantation in rabbits.

AIM: To see if: (i) a large vessel aortocaval vascular patch technique may bring about long-term graft survival after allogeneic uterine transplantation (UTn) in a rabbit model; and (ii) fertility can be achieved following natural mating post-allogeneic UTn. METHODS: Allogeneic uterine cross transplantations were performed in New Zealand white rabbits using an aortocaval macrovascular patch harvested as part of the uterine allograft. Five rabbit recipients received a uterine graft from five unrelated donor rabbits. All female rabbits were unrelated and were of proven fertility with at least one previous litter each. Tacrolimus was administrated for immunosuppression post-transplant. Natural mating was attempted if long-term survival had been achieved. The main outcome measures were: (i) long-term recipient survival; (ii) long-term adequate uterine perfusion; and (iii) successful pregnancy post-UTn. RESULTS: All five recipient animals survived the surgery with satisfactory immediate postoperative recovery. Recipients 1, 2 and 4 died within the first 4 postoperative days. Both long-term survivors failed to conceive following introduction of a proven male breeder despite evidence of mating. Necropsy at 9 and 11 months showed a lack of patency of uterine cornua at the point of anastomosis, albeit a small uterus in recipient 3 and a reddish brown amorphous material at the site of the transplanted uterus in recipient 5. CONCLUSION: We have demonstrated the feasibility of uterine allotransplantation using a macrovascular patch technique, but could not demonstrate conception because of blocked cornua. To address this, we propose using embryo transfer techniques in order to achieve conception.

Sex- and age-specific effects of nutrition in early gestation and early postnatal life on hypothalamo-pituitary-adrenal axis and sympathoadrenal function in adult sheep.

The early-life environment affects risk of later metabolic disease, including glucose intolerance, insulin resistance and obesity. Changes in hypothalamo-pituitary-adrenal (HPA) axis and sympathoadrenal function may underlie these disorders. To determine consequences of undernutrition in early gestation and/or immediately following weaning on HPA axis and sympathoadrenal function, 2- to 3-year-old Welsh Mountain ewes received 100% (C, n = 39) or 50% nutritional requirements (U, n = 41) from 1-31 days gestation, and 100% thereafter. From weaning (12 weeks) to 25 weeks of age, male and female offspring were then either fed ad libitum (CC, n = 22; UC, n = 19) or were undernourished (CU, n = 17; UU, n = 22) such that body weight was reduced to 85% of their individual target, based on a growth trajectory calculated from weights taken between birth and 12 weeks. From 25 weeks, ad libitum feeding was restored for all offspring. At 1.5 and 2.5 years, adrenocorticotropic hormone (ACTH) and cortisol concentrations were measured at baseline and in response to corticotropin-releasing factor (CRF) (0.5 microg kg(1)) plus arginine vasopressin (AVP) (0.1 microg kg(1)). At 2.5 years, HPA axis and sympathoadrenal (catecholamine) responses to a transport and isolation stress test were also measured. In females, post-weaning undernutrition reduced pituitary output (ACTH) but increased adrenocortical responsiveness (cortisol:ACTH area under curve) during CRF/AVP challenge at 1.5 years and increased adrenomedullary output (adrenaline) to stress at 2.5 years. In males, cortisol responses to stress at 2.5 years were reduced in those with slower growth rates from 12 to 25 weeks. Early gestation undernutrition was associated with increased adrenocortical output in 2.5-year-old females only. Pituitary and adrenal responses were also related to adult body composition. Thus, poor growth in the post-weaning period induced by nutrient restriction has sex- and age-specific effects on HPA and sympathoadrenal function. With altered glucose tolerance previously reported in this model, this may have long-term detrimental effects on metabolic homeostasis and cardiovascular function.

The late gestation fetal cardiovascular response to hypoglycaemia is modified by prior peri-implantation undernutrition in sheep.

Undernourished late gestation fetuses display asymmetric growth restriction, suggestive of a redistribution of nutritional resources. The modification of fetal organ blood supply in response to acute hypoxia is well characterized, but it is not known whether similar responses occur in response to acute reductions in nutrition, or if such late gestation responses can be influenced by early gestation nutrition. In pregnant sheep, total nutrient requirements were restricted during the peri-implantation period (PI40, 40%; PI50, 50% of total, days 1-31) or in late gestation (L, 50% total, days 104-postmortem). Control animals were fed 100% nutrient requirements. Fetal organ blood flows were measured at baseline, and during acute fetal hypoglycaemia induced by maternal insulin infusion at 125 dGA. Baseline heart rate was increased in PI40 fetuses. During hypoglycaemia, an initial rise in fetal heart rate was followed by a slower fall. Fetal femoral artery blood flow decreased, and adrenal blood flow and femoral vascular resistance increased in all fetuses during hypoglycaemia. These changes were accompanied by increased fetal plasma adrenaline and cortisol, and reduced plasma insulin levels. The maximum femoral artery blood flow response to hypoglycaemia occurred earlier in PI50 and PI40 compared with control fetuses. The late gestation fetal cardiovascular response to acute hypoglycaemia was consistent with a redistribution of combined ventricular output away from the periphery and towards central organs. One element of the peripheral vascular response was modified by peri-implantation nutrient restriction, indicating that nutritional challenges early in gestation can have an enduring impact on cardiovascular control.

Effects of pre- and periconceptional undernutrition on arterial function in adult female sheep are vascular bed dependent.

The nutritional environment during development and even prior to conception may contribute to cardiovascular risk. In mature adult female sheep, we investigated the effect of preconceptional and periconceptional maternal nutritional restriction on the vascular reactivity of arteries from four vascular beds supplying the heart, thorax, kidney and hindlimb. Welsh Mountain ewes received 100% of nutrient requirements throughout gestation (control group, C, n = 18), or 50% of nutrient requirements for 30 days prior to conception (preconceptional group, PRE, n = 20) or for 15 days either side of conception (periconceptional group, PERI, n = 31) and 100% thereafter. In 3.5-year-old female offspring, the left anterior descending coronary (LAD), left internal thoracic (LITA), right renal and second and third order femoral arteries were dissected and their reactivity was assessed by organ bath or wire myography. Vasoconstrictor responses were greater in both LAD and LITA from PERI offspring compared with C (P < 0.01), while vasoconstriction was unaffected by maternal diet in arteries from the renal and femoral circulations (P = n.s.). Endothelium-dependent and -independent vasodilatation was attenuated in third order femoral arteries of PRE and PERI groups compared with C (P < 0.05). Endothelium-independent vasodilatation was attenuated in both the LAD and renal arteries in the PERI group compared with C (P < 0.05). These data show that moderate maternal undernutrition either prior to or around conception affects vascular function of adult offspring. The effect depends on the timing of the insult, but also on the vascular bed studied and vessel hierarchy in the vascular tree.

Pelvic vascular prospects for uterine transplantation.

While developing the technique of abdominal radical trachelectomy for conservative cervical cancer management, the vascular supply of the uterus was thoroughly examined. This was a prelude to study the possibility of uterine transplantation where initial concerns were about how uterine artery anastomosis might be achieved and the subsequent function of these vessels in pregnancy. In experiment 1, the uterine arteries in two sows were divided and reanastomosed. At 6 weeks, all sows including control were inseminated. After weaning 3 months after delivery, the sows were killed, and postmortem studies were undertaken. Successful reanastomoses of the uterine arteries were accomplished in both study sows. After insemination, pregnancy proceeded uneventfully, and both sows farrowed normally with average litter sizes. Histopathology of the uterine arteries revealed minimal intimal fibrosis across all anastomotic sites. Uterine artery anastomosis in the porcine model is feasible with subsequent normal vascular function in pregnancy of the anastomosed vessels.

Multispectral imaging of organ viability during uterine transplantation surgery in rabbits and sheep.

Uterine transplantation surgery (UTx) has been proposed as a treatment for permanent absolute uterine factor infertility (AUFI) in the case of the congenital absence or surgical removal of the uterus. Successful surgical attachment of the organ and its associated vasculature is essential for the organ's reperfusion and long-term viability. Spectral imaging techniques have demonstrated the potential for the measurement of hemodynamics in medical applications. These involve the measurement of reflectance spectra by acquiring images of the tissue in different wavebands. Measures of tissue constituents at each pixel can then be extracted from these spectra through modeling of the light­tissue interaction. A multispectral imaging (MSI) laparoscope was used in sheep and rabbit UTx models to study short- and long-term changes in oxygen saturation following surgery. The whole organ was imaged in the donor and recipient animals in parallel with point measurements from a pulse oximeter. Imaging results confirmed the re-establishment of adequate perfusion in the transplanted organ after surgery. Cornual oxygenation trends measured with MSI are consistent with pulse oximeter readings, showing decreased StO2 immediately after anastomosis of the blood vessels. Long-term results show recovery of StO2 to preoperative levels.

Effects of pre-natal and early post-natal undernutrition on adult internal thoracic artery function.

OBJECTIVE: Previous studies in humans and animals have suggested that undernutrition in utero and in early post-natal life may lead to altered vascular function in a number of peripheral arteries. We investigated the effect of pre- and post-natal nutrient restriction on the vascular reactivity of the left internal thoracic artery using a sheep model. METHODS: Welsh mountain ewes were mated and assigned to three dietary groups: (1) 100% of total nutritional requirements (control, n=6); (2) 50% of total nutritional requirements during the first 31 days of gestation (n=6); and (3) 50% nutritional restriction during the first 31 days of gestation, followed by a restriction in the diet of their offspring 12-25 weeks post-natally, designed to produce a 15% reduction in growth trajectory (n=7). The male offspring were sacrificed at 130 weeks; the left internal thoracic artery was mounted onto a wire myograph and the reactivity of the vessel to various agonists measured. RESULTS: The offspring of animals who underwent an early gestation nutrient restriction had a significantly increased basal tone (0.41+/-0.25 vs 6.34+/-1.35, p=0.015) and sensitivity to phenylephrine (log EC(50): -6.23+/-0.04 M vs -5.74+/-0.17 M, p=0.036) as compared with control animals. However, this phenomenon was not seen in animals that underwent both pre- and post-natal nutrient restriction. CONCLUSIONS: Pre-natal undernutrition increases the basal tone and sensitivity of the left internal thoracic artery to phenylephrine. This effect is significantly attenuated by continued undernutrition in early post-natal life. These experiments suggest that in utero and early post-natal undernutrition may be important determinants of graft function in later life.

Clinical evaluation of postpartum vaginal mucus reflects uterine bacterial infection and the immune response in cattle.

Bacteria contaminate the uterus of most dairy cattle after parturition and endometritis causes infertility. An endometritis score can be ascribed based on the vaginal mucus character and odour but it is not clear if the clinical score reflects the number of uterine bacteria or the inflammatory response. The present study tested the hypothesis that clinical evaluation of endometritis reflects the number of bacteria present in the uterus, and the acute phase protein response. Swabs (n = 328) were collected from the uterine lumen of dairy cattle, 21 and 28 days postpartum, vaginal mucus was scored for character and odour, and blood samples collected for acute phase protein measurement. Bacteria were identified following aerobic and anaerobic culture, and the bacterial growth density was scored semi-quantitatively. When bacteria were categorised by their expected pathogenic potential in the uterus, purulent or fetid odour vaginal mucus was associated with the growth density of pathogenic bacteria but not opportunist contaminants. When bacteria were analysed independently, Arcanobacterium pyogenes, Proteus and Fusobacterium necrophorum growth densities were associated with mucopurulent or purulent vaginal mucus. The bacterial growth densities for A. pyogenes, Escherichia coli, non-hemolytic Streptococci, and Mannheimia haemolytica were associated with a fetid mucus odour. Peripheral plasma concentrations of alpha(1)-acid glycoprotein were higher if there was a fetid compared with a normal vaginal mucus odour (1.50 +/- 0.09 mg/mL versus 1.05 +/- 0.02 mg/mL, P < 0.001), but did not differ significantly between vaginal mucus character scores. The evaluation of the character and odour of vaginal mucus reflects the number of bacteria in the uterus, and the acute phase protein response.

Is it feasible to use a large vessel patch with a uterine allograft en bloc for uterine transplantation?

Uterine auto-transplantation in the porcine model using a microvascular anastomotic technique failed because of gradual vessel thromboses. A macrovascular patch harvest with the uterus en bloc is probably less likely to undergo vessel thrombosis. The objective of this study was to assess the feasibility of an aortic/inferior vena caval vascular patch harvest with the uterus en bloc for uterine cross-transplantation purposes. A preserved human cadaver and freshly killed porcine and rabbit cadaveric models were used. The infrarenal aorta, inferior vena cava, common and internal iliac vessels and the uterine arterial and venous tree together with the uterus en bloc were successfully harvested intact as a large vessel patch and graft in both animal and human models. A macrovascular patch for uterine cross-transplantation is technically easy and feasible to harvest en bloc with the uterus in fresh cadaveric donors. This technique precludes live donors and makes cadaveric organ donation essential.

Possibilities for fertility restoration: a new surgical technique.

Previously published work concluded that uterine artery microvascular anastomosis in the porcine model was feasible with subsequent normal vascular function in pregnancy. of the anastomosed vessels. The objective of this study was to assess the feasibility of uterine autotransplantation in a porcine model using microvascular anastomoses. Eight large white/landrace sows of proven fertility were used. A supracervical hysterectomy with or without bilateral salpingo-oophorectomy (BSO) was performed. After 1 hour of cold storage in a transplant solution, the specimen was reintroduced and followed by stepwise vascular reanastomosis. Objective perfusion index measurements suggested adequate uterine perfusion after transplantation. Postoperatively, sow 1 survived to 3 months with no signs of oestrus, and sows 2, 6, and 8 were killed on days 6, 33, and 54, respectively, for pelvic infection. Histopathology of the uterine grafts revealed gradual vessel thromboses. Microvascular reanastomosis is feasible, however, unsuccessful in uterine autotransplantation because of gradual vessel thromboses. The porcine model is highly susceptible to postoperative infection.

The effect of intrauterine administration of estradiol on postpartum uterine involution in cattle.

In cattle, the first postpartum dominant follicle has a predilection for the ovary contralateral to the previously gravid uterine horn. However, the presence of an estradiol-secreting dominant follicle in the ipsilateral ovary is a marker of subsequent fertility, possibly due to a localized effect of ovarian estradiol on uterine involution. The present study tested the hypothesis that estradiol increases the rate of uterine involution when administered into the previously gravid uterine horn around the expected time of selection of the first postpartum dominant follicle. Dairy cows were treated with 10 mg estradiol benzoate (n=15) or saline (n=14) administered through the cervix into the previously gravid uterine horn lumen on Days 7 and 10 postpartum. Uterine involution was monitored by daily transrectal ultrasonography and estimation of peripheral plasma concentrations of PGFM and acute phase proteins, while ovarian function was monitored by ultrasonography and measurement of plasma hormone concentrations. There was no effect of estradiol treatment on the diameter of the previously gravid or nongravid uterine horns, nor on the plasma concentrations of PGFM or acute phase proteins. However, cows in which the first postpartum dominant follicle ovulated during the study period had a smaller diameter of the previously gravid (P<0.01) or nongravid uterine horns (P<0.001) compared with cows in which the follicle regressed. Thus, our hypothesis was not proven, and the opposite pathway of utero-ovarian signaling may be more important during the postpartum period.

Metabolite contents of blood serum and fluid from small and large sized follicles in dromedary camels during the peak and the low breeding seasons.

This study was undertaken to investigate serum and follicular fluid (FF) concentrations of some biochemical metabolites during the low (May to October) and the peak breeding season (November to April) in female camels with small and large follicles. For this purpose, ovaries from 92 female camels aged 3-7 years (young) or 8-15 years (adult) with clinically normal reproductive tract and slaughtered over a 24-month period were collected. Jugular blood samples and FF aspirated from small (5-9 mm) and large (10-20mm) follicles were analyzed for various metabolite concentrations, using the commercial kits. The effect of season, age and follicular size on serum glucose levels was not significant. However, FF glucose concentration in small follicles (136.79+/-4.05 mg/dl) was higher (P<0.05) than that of large follicles (77.09+/-4.31 mg/dl). Serum cholesterol contents were neither affected by the season, nor by age of the animal or the size of the ovarian follicles. The FF cholesterol concentration during the low breeding season (21.08+/-1.11 mg/dl) was higher (P<0.05) than 6.25+/-1.14 mg/dl recorded during the peak breeding season. The serum and FF total protein and albumin concentrations were neither affected by the season, nor by the age of the animal or the size of the ovarian follicles. The FF globulin concentration during the peak breeding season (2.46+/-0.06 g/dl) was higher (P<0.05) than 1.56+/-0.06 g/dl recorded during the low breeding season. Serum and FF activities of AST and ALT did not differ between the two seasons, age groups or follicle classes. Serum triglycerides (56.12+/-1.28 mg/dl) and HDL (45.12+/-0.12 mg/dl) levels during the peak breeding season were higher (P<0.01) than 31.91+/-1.25 and 42.60+/-0.11 mg/dl, respectively, observed during the low breeding season. Serum and FF triglycerides were neither affected by age nor by follicle size. Serum HDL concentration was higher (P<0.05) in adult than young camels. The concentration of HDL in FF was higher (P<0.05) during the peak (41.92+/-0.06 mg/dl) than the low (40.80+/-0.06 mg/dl) breeding season. It was concluded that serum contents of triglycerides and HDL were influenced by the breeding season. Similarly, FF contents of cholesterol, globulin and HDL were influenced by season, while FF glucose contents were influenced by the size of the follicle. However, no correlation could be established between serum and follicular fluid contents of various biochemical metabolites included in the study.

The effect of maternal undernutrition in early gestation on gestation length and fetal and postnatal growth in sheep.

In utero undernutrition in humans may result in cardiovascular (CV), metabolic, and growth adaptations. In sheep, maternal nutrient restriction during pregnancy, without effects on fetal or birth weight, results in altered CV control in the offspring. Adjustment of gestation length after undernutrition could be a strategy to enhance postnatal health/survival. The aim of this study was to determine in sheep the effect of a 50% reduction in maternal nutrient intake [undernutrition group (U) versus 100%, control group (C)] during 1-31 d of gestation (dGA) on gestation length and offspring size. By 28 dGA, U ewes had gained less weight than C, and twin-bearing ewes had gained less weight than singleton-bearing ewes regardless of group (p<0.05). In different-sex twin pairs, maternal undernutrition resulted in longer gestation compared with C (146.5+/-0.6 versus 144.6+/-0.6 d, p<0.05). Increased weight gain by weaning (20.8+/-0.8 versus 17.9+/-0.8 kg, p<0.05) was observed in U male twins. These findings suggest that the strategy (i.e. growth rate or length of time in utero) adopted by the fetus to enhance immediate survival depends on offspring number and sex. This is likely to reflect the degree of constraint imposed on the fetus.

Nutritional challenges during development induce sex-specific changes in glucose homeostasis in the adult sheep.

The early-life environment has implications for risk of adult-onset diseases, such as glucose intolerance, insulin insensitivity, and obesity, effects that may occur with or without reduced birth weight. We determined the consequences of nutrient restriction in early gestation and early postnatal life and their interactions on postnatal growth, body composition, and glucose handling. Ewes received 100% (C, n = 39) or 50% nutritional requirements (U, n = 41) from 1 to 31 days gestation and 100% thereafter. Male and female offspring (singleton/twin) from C and U ewes were then fed either ad libitum (CC n = 22, UC n = 19) or to reduce body weight to 85% of target from 12 to 25 wk of age (CU n = 17, UU n = 22) and ad libitum thereafter. At 1.5 and 2.5 yr, glucose handling was determined by area under the curve (AUC) for glucose and insulin concentrations following intravenous glucose (0.5 g/kg body wt). Insulin sensitivity was determined at 2.5 yr following intravenous insulin (0.5 IU/kg). In females, postnatal undernutrition reduced (P < 0.05) glucose AUC at both ages, regardless of prenatal nutrition. Postnatal undernutrition did not affect insulin secretion in females but enhanced insulin-induced glucose disappearance in singletons. Poor early postnatal growth was associated with increased fat in females. In males, glucose tolerance was unaffected by undernutrition despite changes in insulin AUC dependent on age, treatment, and single/twin birth. Nutrition in early postnatal life has long-lasting, sex-specific effects on glucose handling in sheep, likely due, in females, to enhanced insulin sensitivity. Improved glucose utilization may aid weight recovery but have negative implications for glucose homeostasis and body composition over the longer term.

Mismatched pre- and postnatal nutrition leads to cardiovascular dysfunction and altered renal function in adulthood.

The early life environment has long-term implications for the risk of developing cardiovascular (CV) disease in adulthood. Fetal responses to changes in maternal nutrition may be of immediate benefit to the fetus, but the long-term effects of these adaptations may prove detrimental if nutrition in postnatal life does not match that predicted by the fetus on the basis of its prenatal environment. We tested this predictive adaptive response hypothesis with respect to CV function in sheep. We observed that a mismatch between pre- and postnatal nutrient environments induced an altered CV function in adult male sheep that was not seen when environments were similar. Sheep that received postnatal undernutrition alone had altered growth, CV function, and basal hypothalamo-pituitary-adrenal axis activity in adulthood. Prenatal undernutrition induced greater weight gain by weaning compared with the prenatal control diet, which may provide a reserve in the face of a predicted poor diet in later life. In an adequate postnatal nutrient environment (i.e., relatively mismatched), these offspring exhibited cardiac hypertrophy and altered CV function in adulthood. These data support the concept that adult CV function can be determined by developmental responses to intrauterine nutrition made in expectation of the postnatal nutritional environment, and that if these predictions are not met, the adult may be maladapted and at greater risk of CV disease. Our findings have substantial implications for devising strategies to reduce the impact of a mismatch in nutrition levels in humans undergoing rapid socio-economic transitions in both developing and developed societies.

Effect of the regressing corpus luteum of pregnancy on ovarian folliculogenesis after parturition in cattle.

In cattle, the first postpartum dominant follicle has a predilection for the ovary contralateral to the previously gravid uterine horn, possibly due to a local inhibitory effect of the regressing corpus luteum of pregnancy in the ipsilateral ovary. The aim of the present study was to test the hypothesis that the regressing corpus luteum of pregnancy suppresses folliculogenesis in the ipsilateral ovary after parturition. Dairy cows were treated with prostaglandin F2alpha between 190 and 220 days of gestation to cause luteolysis without inducing parturition (n = 14) or were untreated controls (n = 32). Follicular growth and function were monitored by daily transrectal ultrasonography and collection of plasma samples for estimation of FSH, estradiol, and progesterone concentrations. The proportion of first dominant follicles in the ipsilateral ovary was similar for treated and control animals (4/14 vs. 8/32), as was the time interval between calving and establishment of a dominant follicle (mean +/- SEM, 10.1 +/- 0.4 vs. 10.7 +/- 0.5 days). Furthermore, no significant effect of treatment on dominant follicle growth or function was found as determined by plasma hormone concentrations. Although greater folliculogenesis was found in the ovary contralateral to the previously gravid uterine horn, once the location of the future first dominant follicle was selected, the timing of events was independent of location. We suggest that the corpus luteum of pregnancy does not have a local effect on postpartum ovarian folliculogenesis and that, instead, an effect of the previously gravid uterine horn shortly after parturition should be considered.

Effect of nutritional restriction in early pregnancy on isolated femoral artery function in mid-gestation fetal sheep.

Unbalanced maternal nutrition affects fetal endocrine and cardiovascular systems, sometimes accompanied by changes in growth, although this is usually in late gestation. We determined the effect of moderate restriction for the first half of gestation of maternal dietary protein, or of total calorific intake on isolated resistance artery function of mid-gestation fetal sheep. Welsh Mountain ewes were nutritionally restricted by 30 % of the recommended nutrient intake (globally restricted) or 30 % of the recommended protein intake (protein-restricted), compared to control ewes fed 100 % of recommended nutrient intake, for ~12 days prior to conception and for the subsequent 70 days of gestation. At mid-gestation, fetal and placental weights were similar in all dietary groups. In isolated femoral arteries, the response curve to noradrenaline was reduced in protein-restricted group fetuses (P < 0.05). Maximal relaxation (P < 0.01) and sensitivity (P < 0.05) to acetylcholine were markedly reduced in protein-restricted group fetuses, and to a smaller extent in globally restricted group fetuses (response curve, P < 0.05). The dilator response (P < 0.05) and sensitivity (P < 0.05) to the alpha2 agonist UK14304 was lower in protein-, but not in globally restricted group fetuses. The response (P < 0.05) and sensitivity (P < 0.05) to the nitric oxide donor sodium nitroprusside were reduced in protein-restricted group fetuses compared to controls. Our data show that dietary imbalance, in particular restricted protein, of the ewe can produce blunting of endothelial-dependent and -independent relaxation in systemic arteries from the mid-gestation fetus. These changes may precede perturbed late-gestation fetal and postnatal cardiovascular control.