The challenge of establishing, growing and sustaining a large biobank: a personal perspective.

Laboratory medicine professionals have a unique understanding of the wealth that biological samples bring to clinical research, and of the need for quality standards for the collection, transportation, storage and analytical phases. The expertise of laboratory physicians and scientists also adds value to the interpretation and publication of the results of clinical research studies. This is an account of the evolution of over thirty five years of the Biobank/Clinical Research Clinical Trials Laboratory at one Canadian health sciences centre. The logistical, financial, and quality management challenges are presented in growing from a small-scale facility to one that now stores three million well-characterized samples from more than seventy countries, representing five continents and five major ethnic groups. This is an account of a journey, it is not intended as a guide as to how to create an 'ideal' biobank. Collaboration, collegiality, consistency, creativity and clinical collaborators, are the keys to progress, but there must first be a vision, one that can expand to embrace new opportunities.

Morphology of pig uterine subepithelial capillaries after topical and systemic oestrogen treatment.

The ultrastructure of subepithelial capillaries in pig endometrium was studied after intrauterine and systemic oestradiol treatment. Gilts were killed on day 13 or 19 (n = 4 per day per treatment) after surgical introduction of Silastic beads containing either oestradiol or cholesterol into the uterine lumina on day 10 after oestrus. An additional group of gilts was injected i.m. with 5 mg oestradiol valerate on days 11-15 of the oestrous cycle and killed on day 13 or day 19 (n = 4 per day). Light and electron microscope studies of endometrial samples revealed that cholesterol beads did not appear to affect subepithelial capillary structure. Both intrauterine and systemic treatment of gilts with oestradiol were associated with regional modification of the capillary wall involving attenuation and fenestration of the face directly underlying the uterine epithelium, accumulation of caveolae in the opposite face of the capillary, and the occurrence of discontinuous, multilayered capillary basal laminae. The similarity of these structural changes to those observed in capillaries at the time of embryonic attachment suggests that oestrogens of blastocyst origin may function to modify capillary morphology and possibly facilitate increased transcapillary traffic during the establishment of pregnancy.

Effects of topical and systemic estrogen on morphology of porcine uterine luminal epithelia.

Silastic beads containing either estradiol-17 beta or cholesterol were surgically introduced into the uterine lumina of gifts on Day 10 postestrus, and the animals were killed on Day 13 or 19 (n = 4/day/treatment). An additional group of gifts were injected i.m. with 5 mg estradiol valerate on Days 11-15 of the estrous cycle and were killed on Day 13 and Day 19 (n = 4/day). Endometrial samples were collected and prepared for light and electron microscopic study. Cholesterol beads did not appear to affect endometrial structure. Both intrauterine and systemic treatment of gilts with estradiol induced folding along the mesometrial region of the uterus similar to that which occurs during early pregnancy. The uterine luminal epithelium of animals exposed to both exogenous hormone treatments exhibited morphological features similar to those of this layer during early pregnancy: evidence of increased synthetic and secretory activity, decreased incidence of cell degeneration, accumulation of glycogen and dense inclusions, variation in nuclear position, and elaboration of a thick fibrous glycocalyx.

Microscopic examination of porcine conceptus-maternal interface between days 10 and 19 of pregnancy.

Conceptus-maternal interactions in the pig were examined at days 10, 13, 16, and 19 of pregnancy. Contact between the chorion and uterine epithelium was not preserved on day 10, but extensive contact occurred in 3 of 5 pregnant gilts on day 13 and was related to localized differentiation of the uterine epithelium. Attachment events occurred earlier and more rapidly than previously reported, commencing in the region of the embryonic disc and progressing toward the extremities of the chorionic vesicle, with stages from apposition through attachment occurring simultaneously along a conceptus at day 13 and thereafter. Apical protuberances on maternal epithelial cells and interposing blunt chorionic processes were evident at sites of early maternal-conceptus interaction, but were reduced at regions exhibiting microvillous interdigitation. Placentation in the pig represents a developmental process consisting of a continuum of sequential events which occur over a broad time frame rather than at specific discrete times.

Effect of indomethacin and prostaglandin E2 on structural differentiation of rat endometrium during artificially induced decidualization.

Morphological differentiation of uterine stromal and luminal epithelial cells was studied in steroid-injected ovariectomized rats following unilateral intrauterine instillation of sesame oil, phosphate-buffered saline containing gelatin (PBSG), PBSG + indomethacin (IM; an inhibitor of prostaglandin synthesis), or PBSG + IM + prostaglandin E2 (PGE2). The latter two treatments were preceded by a subcutaneous injection of IM. Uteri were examined by light and electron microscopy at intervals between 8 and 120 hr (n = 4/treatment/time). Differentiation began in the periluminal antimesometrial region and progressed peripherally and towards the mesometrial aspect in all groups. Structural features and timing of differentiation were similar for oil-injected and PBSG-infused uteri. Administration of IM inhibited the onset of the decidual cell reaction and had deleterious effects on the luminal epithelium. Inclusion of PGE2 in the instillate accelerated stromal cell differentiation and overcame the inhibitory effect of IM. The results implicate prostaglandins, particularly PGE2, in endometrial transformation during decidualization.

Structural changes in the luminal epithelium of the porcine uterus between days 10 and 19 of the estrous cycle.

Light and electron microscopy were used to study the morphology of uterine luminal epithelium from 4 or 5 gilts slaughtered on each of days 10, 13, 16, and 19 of the estrous cycle. Ultrastructural evidence indicated that metabolic activity and accumulation of glycogen by the uterine epithelium increased between days 10 and 16 of the estrous cycle. This phase of high synthetic activity had been terminated by day 19, as evidenced by the reduction or absence of glycogen deposits and decreased incidence of organelles associated with synthetic activity. Diffuse degeneration of epithelial cells occurred throughout the period of study but was maximal between days 16 and 19. Mitotic activity indicated that cell replacement also occurred between day 16 and day 19.

Autofluorescence of the porcine endometrium during early pregnancy.

Uteri recovered from cyclic gilts (n = 5) on Days 15-19 and pregnant animals (n = 34) on Days 13-40 were opened and examined under UV light. A line of greenish fluorescence was present in the mesometrial region in contact with embryonic membranes at Day 13. Small patches of reddish fluorescence subsequently appeared on the uterine mucosa near the embryonic disc, and these increased in intensity and size until they encompassed the entire area of contact between each conceptus and the endometrium, for lengths of about 20 cm, by Day 29. Fluorescence then diminished gradually and was almost totally absent by Day 40. Three additional gilts were unilaterally hysterectomized on Day 15 and treated with Evans blue dye 10 min before removal of the second uterine horn. Both horns were opened and compared under UV light, but no difference in the pattern of fluorescence could be detected. All fluorescence was associated with uterine rather than conceptus tissues. The occurrence of autofluorescence in uteri of pregnant pigs precludes use of Evans blue dye as an indicator of vascular permeability.

Morphological evidence for increased uterine vascular permeability at the time of embryonic attachment in the pig.

The morphology of subepithelial capillaries was studied in the endometrium of cyclic and pregnant gilts between Days 10 and 19 after estrus to determine whether evidence of increased vascular permeability accompanied embryonic attachment. No differences in the structure of the vessels were observed between Day 10 of the cycle and Day 10 of gestation, but striking regional modification had occurred by Day 13 of pregnancy. The portion of the capillary wall that was most proximal to the uterine epithelium was most attenuated and contained fenestrated segments, and caveolae had accumulated within the thicker distal face. As attachment progressed to Day 19 of pregnancy, thin fenestrated regions were even more extensive and the subepithelial capillaries were located in closer proximity to the uterine epithelium. In addition, modification of the basal lamina resulted in a discontinuous, thickened and multilayered appearance. These findings indicate a temporal association between morphological evidence of increased uterine vascular permeability and a close physical association between the uterine mucosa and trophoblast of elongated porcine blastocysts.

Endometrial prostaglandin E2-binding sites in the pig: characterization and changes during the estrous cycle and early pregnancy.

Because prostaglandins (PGs) are thought to mediate, in part, endometrial responses at implantation in a number of species and because pig conceptuses are capable of producing PGs, we have characterized the binding of [3H]PGE2 to endometrium obtained from pigs during the estrous cycle and early pregnancy. Binding of [3H]PGE2 to a membrane preparation obtained from endometrium was temperature- and pH-dependent, and reversible. Pretreatment of the membrane preparation with proteolytic enzymes, phospholipase A2, or heat reduced subsequent specific binding of [3H]PGE2. PGE1 and PGE2 competed equally for binding, whereas relative cross-reactivity for other prostanoids and compounds tested was less than 5%. Scatchard analysis of specific binding suggested that two classes of binding sites were present, a high-affinity binding site with an apparent dissociation constant (Kd) of approximately equal to 6 pM, and a lower-affinity site with an apparent Kd of approximately equal to 3 nM. For the high-affinity binding site, estimates of apparent Kd did not differ between days of the estrous cycle, but maximum binding capacity did change, being lowest on Day 10 and highest on Days 0-5 of the cycle. For the lower-affinity binding site, estimates of the apparent Kd differed, being lowest on Day 10 and highest on Day 15; numbers of binding sites were lowest on Day 5 and highest on Day 15. Estimates of these variables on Day 15 of pregnancy did not differ significantly from Day 15 of the cycle. These data are consistent with the presence of PGE receptors within the endometrium of the pig.

Increased uterine vascular permeability at the time of embryonic attachment in the pig.

The temporal relationship between embryonic attachment and endometrial vascular permeability was investigated in the gilt. Light and electron microscopy failed to reveal structural differences between Day 10 cycling and pregnant maternal epithelia, including evidence of blastocyst contact. Chorionic adhesion was preserved at mesometrial regions in 3 of 5 Day 13 pregnant animals and appeared to be related to localized differentiation of the underlying maternal epithelium. In order to study uterine vascular permeability, 44 gilts between Days 11 and 19 of the cycle and pregnancy were injected i.v. with a 0.5% solution of Evans Blue (2.5 ml/kg body weight). Examination of excised uteri under ultraviolet light revealed a well-defined zone of endometrial fluorescence corresponding to extravascular content of the dye. Exclusive to pregnant gilts, this response appeared in conjunction with blastocyst elongation at Day 12, and was consistently confined to areas of embryonic membrane contact thereafter. The changes in endometrial morphology and vascular permeability suggest involvement of some embryonic factor(s) acting in a localized manner. Increased histotrophe production is probably facilitated by the flux of plasma constituents to maternal epithelial cells. Coincidence of increased uterine vascular permeability at the site of attachment with elevated blood flow would enhance transport of nutrients toward the conceptus and allow access of blastocyst-induced products to the maternal circulation.