Placental vascular development (angiogenesis) is critical for placental function and thus for normal embryonic/fetal growth and development. Specific environmental factors or use of assisted reproductive techniques may result in poor placental angiogenesis, which may contribute to embryonic losses and/or fetal growth retardation. Uterine tissues were collected on days 14, 16, 18, 20, 22, 24, 26, 28, and 30 after mating and on day 10 after estrus (nonpregnant controls) to determine vascular development and expression of several factors involved in the regulation of angiogenesis in the endometrium. Compared with controls, several measurements of endometrial vascularity increased (P<0.001) including vascular labeling index (LI; proportion of proliferating cells), the tissue area occupied by capillaries, area per capillary (capillary size), total capillary circumference per unit of tissue area, and expression of factor VIII (marker of endothelial cells), but capillary number decreased (P<0.001). Compared with controls, mRNA for placental growth factor, vascular endothelial growth factor receptors, angiopoietins (ANGPT) 1 and 2, ANGPT receptor TEK, endothelial nitric oxide synthase, and hypoxia-inducible factor 1α increased (P<0.05) during early pregnancy. Vascular LI was positively correlated (P<0.05) with several measurements of vascularity and with mRNA expression of angiogenic factors. These data indicate that endometrial angiogenesis, manifested by increased vascularity and increased expression of several factors involved in the regulation of angiogenesis, is initiated very early in pregnancy. This more complete description of early placental angiogenesis may provide the foundation for determining whether placental vascular development is altered in compromised pregnancies.

To characterize early fetal placental development, gravid uterine tissues were collected from pregnant ewes every other day from day 16 to 30 after mating. Determination of 1) cell proliferation was based on Ki67 protein immunodetection; 2) global methylation was based on 5-methyl-cytosine (5mC) expression and mRNA expression for DNA methyltransferases (DNMTs) 1, 3a, and 3b; and 3) vascular development was based on smooth muscle cell actin immunolocalization and on mRNA expression of several factors involved in the regulation of angiogenesis in fetal membranes (FMs). Throughout early pregnancy, the labeling index (proportion of proliferating cells) was very high (21%) and did not change. Expression of 5mC and mRNA for DNMT3b decreased, but mRNA for DNMT1 and 3a increased. Blood vessels were detected in FM on days 18–30 of pregnancy, and their number per tissue area did not change. The patterns of mRNA expression for placental growth factor, vascular endothelial growth factor, and their receptors FLT1 and KDR; angiopoietins 1 and 2 and their receptor TEK; endothelial nitric oxide synthase and the NO receptor GUCY13B; and hypoxia inducing factor 1 α changed in FM during early pregnancy. These data demonstrate high cellular proliferation rates, and changes in global methylation and mRNA expression of factors involved in the regulation of DNA methylation and angiogenesis in FM during early pregnancy. This description of cellular and molecular changes in FM during early pregnancy will provide the foundation for determining the basis of altered placental development in pregnancies compromised by environmental, genetic, or other factors.

Nutrition has been shown to influence several reproductive functions, including hormone production, oocyte competence and fertilization, and early embryonic development. To determine the effects of maternal diet on in vitro fertilization (IVF) and early embryonic development, ewes (n = 18; 47.0 ± 1.5 kg of initial BW) were divided into control and underfed (60% of control) nutritional planes for 8 wk before oocyte collection. Pelleted diets containing 2.4 Mcal of ME/kg and 13% CP (DM basis) were fed once daily. During the first 4-wk acclimation phase, control and underfed ewes were fed 1,000 and 600 g/d, respectively. From wk 4 to 8, control (adequate) ewes were fed to maintain BW and offered 720 g/d, whereas underfed ewes received 432 g/d (60% restricted). Synchronization of estrus was performed using progestagen sponges for 14 d. Follicular development was induced by twice daily injections of FSH on d 13 (5 units/injection) and 14 (4 units/injection) of the estrous cycle. Oocytes were collected from all visible follicles on d 15 of the estrous cycle. After IVF, the proportion of developing embryos was evaluated throughout an 8-d culture period. Under-nutrition decreased (P < 0.006) the rate of cleavage, number of blastocysts per ewe, and rate of blastocyst formation (from 79 to 64%; from 3.3 to 0.8; and from 31 to 8%, respectively). However, the number of visible follicles, total number of oocytes, number of healthy oocytes, percentage of healthy oocytes, number of cleaved oocytes, and morula formation per ewe were similar for control and underfed ewes. These data indicate that undernutrition of donor ewes, resulting in lower BW and BCS, has a negative effect on oocyte quality, which results in lower rates of cleavage and blastocyst formation.

Utero-placental growth and vascular development are critical for pregnancy establishment that may be altered by various factors including assisted reproductive technologies (ART), nutrition, or others, leading to compromised pregnancy. We hypothesized that placental vascularization and expression of angiogenic factors are altered early in pregnancies after transfer of embryos created using selected ART methods. Pregnancies were achieved through natural mating (NAT), or transfer of embryos from NAT (NAT-ET), or IVF or in vitro activation (IVA). Placental tissues were collected on day 22 of pregnancy. In maternal caruncles (CAR), vascular cell proliferation was less (P<0.05) for IVA than other groups. Compared with NAT, density of blood vessels was less (P<0.05) for IVF and IVA in fetal membranes (FM) and for NAT-ET, IVF, and IVA in CAR. In FM, mRNA expression was decreased (P<0.01–0.08) in NAT-ET, IVF, and IVA compared with NAT for vascular endothelial growth factor (VEGF) and its receptor FLT1, placental growth factor (PGF), neuropilin 1 (NP1) and NP2, angiopoietin 1 (ANGPT1) and ANGPT2, endothelial nitric oxide synthase 3 (NOS3), hypoxia-inducible factor 1A (HIF1A), fibroblast growth factor 2 (FGF2), and its receptor FGFR2. In CAR, mRNA expression was decreased (P<0.01–0.05) in NAT-ET, IVF, and IVA compared with NAT for VEGF, FLT1, PGF, ANGPT1, and TEK. Decreased mRNA expression for 12 of 14 angiogenic factors across FM and CAR in NAT-ET, IVF, and IVA pregnancies was associated with reduced placental vascular development, which would lead to poor placental function and compromised fetal and placental growth and development.

Background: The objective of this study was to perform complex characterization of cryopreserved and then autotransplanted ovaries including determination of the ability to respond to in vivo follicle stimulating hormone (FSH)-treatment, fertilizability of retrieved oocytes, and morphology, vascularization, cellular proliferation and apoptosis in sheep. Methods: Mature crossbred ewes were divided into two groups; an intact (control) group (n = 4), and autotransplanted group (n = 4) in which oophorectomy was performed laparoscopically and ovaries with intact vascular pedicles frozen, thawed and transplanted back into the same animal at a different site. Approximately five months after autotransplantation, estrus was synchronized, ewes were treated with FSH, and ovaries were collected. For all ovaries, number of visible follicles was determined, and collected cumulus oocyte complexes (COC) were matured and fertilized in vitro. Remaining ovarian tissues were fixed for evaluation of morphology, expression of factor VIII (marker of endothelial cells), vascular endothelial growth factor (VEGF; expressed by pericytes and smooth muscle cells), and smooth muscle cell actin (SMCA; marker of pericytes and smooth muscle cells), and cellular proliferation and apoptosis. Two fully functional ovaries were collected from each control ewe (total 8 ovaries). Results: Out of eight autotransplanted ovaries, a total of two ovaries with developing follicles were found. Control ewes had 10.6 +/- 2.7 follicles/ovary, oocytes were in vitro fertilized and developed to the blastocyst stage. One autotransplanted ewe had 4 visible follicles from which 3 COC were collected, but none of them was fertilized. The morphology of autotransplanted and control ovaries was similar. In control and autotransplanted ovaries, primordial, primary, secondary, antral and preovulatory follicles were found along with fully functional vascularization which was manifested by expression of factor VIII, VEGF and SMCA. Proliferating cells were detected in follicles, and the rate of apoptosis was minimal in ovaries of control and autotransplanted ovaries. Conclusion: These data demonstrate successful autotransplantation of a portion of frozen/thawed ovaries manifested by restoration of selected ovarian function including in vitro maturation of collected oocytes, presence of follicles from several stages of folliculogenesis and blood vessels expressing specific markers of vascularization, and proliferation and apoptosis of ovarian cells. Thus, heterotopic autotransplantation of a whole frozen/thawed ovary allows for development of preovulatory follicles, oocyte growth, and for restoration of vascularization and cellular function. However, additional improvements are required to enhance the efficiency of autotransplantation of frozen/thawed ovaries to produce more oocytes.