Morphometry of implantation in sheep. I. Trophoblast attachment, modification of the uterine lining, conceptus size and embryo location

The degree of trophoblastic attachment, modification of the uterine lining epithelium during implantation, possible differences in size of the conceptus, and preferential zones of nidation were evaluated. Eight ewes at 14 (n=3), 17 (n=2), 20 (n=1), and 24 (n=2) d of gestation were used, and 6 to 8 equidistant samples of either uterine o uterine plus embryonic membranes tissues were taken from each uterine horn for histomorphometric analysis. Trophoblastic attachment varied from 1.5 to 100% at 14 and 24 d of gestation, respectively. Modification of the uterine lining epithelium ranged from 0% at 14 d to 90% at 24 d. The conceptuses occupied a variable area of the uterine cavity; 15.1% at 14 d, 100% at 17 d, 70.6% at 20 d and 100% at 24 d of gestation. All the embryos were located caudal to the center of the uterine horn. Attachment of the trophoblast and modification of the uterine lining epithelium occurred in the area surrounding the embryos. Implantation was a gradual and long-lasting process which commenced near the embryo and extended peripherally. Morphological variations need to be considered when evaluating embryonic viability.     

Effects of indomethacin on uterine capillary permeability and blastocyst development in rabbits

Increased capillary permeability at implantation sites was demonstrated in rabbits by extravasation of intravascular blue dye on day 7 of pregnancy. Subcutaneous administration of indomethacin (Id, 8 mg/kg twice daily) on days 4–6 of pregnancy inhibited this uterine blueing response and appeared to reduce the size of implantation swellings. To test the latter observation blastocyst diameter and development of the embryonic disk were assessed at 144 hr . In females receiving indomethacin at the dose level which inhibited uterine blueing, blastocysts were significantly smaller than those from control females. Developmental staging of embryonic disks revealed only slight differences between the smaller (Id-treated) blastocysts and control blastocysts. No effect of Id was seen on ovarian function as judged by luteal weights and plasma progesterone and estradiol levels. Since the major biological effects of indomethacin are due to its inhibition of prostaglandin synthesis, it appears that prostaglandins may play a role in the uterine response to blastocyst stimulation and in the expansion of blastocysts in the rabbit.     

Chorionic gonadotrophin and embryo-maternal recognition during the peri-implantation period in primates

Genes for chorionic gonadotrophin (CG) are transcribed by the 16-cell embryo stage in humans, but there is no clear evidence of CG secretion as a bioactive dimer before attachment and trophoblast outgrowth stages of implantation. The studies summarized question the timing of CG expression and secretion, the possible roles of CG for intraembryonic differentiation and at the implantation site, and the recognition of this primate embryo-derived signal in support of the corpus luteum. The data suggest that the implantation window in primates may be broader than in non-primate species, where a closer synchrony between embryonic, tubal and uterine events appears to be necessary for embryonic survival. Some preliminary data concerning an association between peripheral thrombocytopenia, ovarian inhibin secretion and peri-implantation stages of embryo development indicate that an unknown embryonic signal may be secreted before bioactive CG can be detected.     

Uterine secretion of prostaglandin F2 alpha in response to oxytocin in ewes: changes during the estrous cycle and early pregnancy.

Experiment 1 was conducted to determine when the ovine uterus develops the ability to secrete prostaglandin F2 alpha (PGF2 alpha) in response to oxytocin and how development is affected by pregnancy. Pregnant and nonpregnant ewes received an injection of oxytocin (10 IU, i.v.) on Day 10, 13, or 16 postestrus. Jugular venous blood samples were collected for 2 h after injection for quantification of 13,14-dihydro-15-keto-PGF2 alpha (PGFM). In nonpregnant ewes, concentrations of PGFM increased following oxytocin on Day 16 but not on Day 10 or 13. Concentrations of PGFM did not increase following treatment on Day 10, 13, or 16 in pregnant ewes. Therefore, the ability of oxytocin to induce uterine secretion of PGF2 alpha develops after Day 13 in nonpregnant but not in pregnant ewes. Experiment 2 was conducted to precisely define when uterine secretory responsiveness to oxytocin develops. Pregnant and nonpregnant ewes received oxytocin on Day 12, 13, 14, or 15. In nonpregnant ewes, concentrations of PGFM increased following treatment on Days 14 and 15, but not earlier. Peripheral concentrations of progesterone showed that uterine secretory responsiveness to oxytocin developed prior to the onset of luteal regression. As in experiment 1, the increase in concentrations of PGFM following administration of oxytocin was much lower in pregnant than in nonpregnant ewes; however, some pregnant ewes did respond to oxytocin with an increase in PGFM. In experiment 3, pregnant ewes received an injection of oxytocin on Day 18, 24, or 30 postmating. Concentrations of PGFM increased following oxytocin on Days 18 and 24. The conceptus appears to delay and attenuate the development of uterine secretory responsiveness to oxytocin.     

Delayed uterine fluid clearance and reduced uterine perfusion in bitches with endometrial hyperplasia and clinical management with postmating antibiotic

In many species a transient uterine inflammatory response follows mating and is proposed to remove excess spermatozoa, bacteria, and other contaminants from the uterus. Similar events have been documented in the bitch involving increased uterine contractions, polymorphonuclear neutrophil influx and uterine artery vasodilation. Some healthy bitches with endometrial hyperplasia have increased numbers of uterine luminal polymorphonuclear neutrophils after mating and reduced fertility; it is purported that this represents a presumed postmating endometritis. This study used B-mode and Doppler ultrasonography at the time of mating to measure uterine contractions, clearance of ejaculated fluid, and uterine artery velocity in normal bitches and those with endometrial hyperplasia. Mating resulted in an increase in the number of uterine contractions, although fewer mating-induced contractions were noted in bitches with endometrial hyperplasia. Interestingly, uterine fluid cleared significantly more slowly after mating from the bitches with endometrial hyperplasia than the normal bitches (P = 0.01). In a further study, Doppler ultrasonography showed that in normal bitches there was a significant increase in uterine artery blood velocity (P = 0.04) and a decrease in the resistance index after mating (P = 0.04), indicating vasodilation. In bitches with endometrial hyperplasia the baseline resistance index was significantly higher than normal bitches (P = 0.05), and furthermore, although there was a significant decrease in resistance index after mating, in the bitches with endometrial hyperplasia this was of a smaller magnitude that in normal bitches. These findings indicate lower baseline uterine perfusion, and a blunted vasodilation response to mating in bitches with endometrial hyperplasia. Short-duration postmating administration of systemic antibiotic increased pregnancy rates in bitches with endometrial hyperplasia (P < 0.01). Litter sizes in bitches with endometrial hyperplasia were lower than those of normal bitches both before and after treatment with postmating antibiotic (P = 0.04 and < 0.01, respectively). Mating-induced endometritis in bitches with endometrial hyperplasia appears to affect fertility by reducing the uterine vasodilatory response to mating and delaying clearance of uterine fluid as a result of decreased uterine contractions but the effect can be ameliorated in part by the postmating administration of antibiotic.     

Effect of cyclooxygenase on "window of implantation" in mouse

The major determinants of uterine receptivity are the ovarian progesterone and estrogen hormones, respectively. Different prostaglandins (PGs) have been elucidated in reproduction and also in this process of implantation in various ways. The blastocyst undergoes implantation on the uterine epithelium in defined hormone prepared period known as "implantation window". However, any definitive role of PGs in the window of receptivity remains elusive. It is demonstrated herein that selective COX1 inhibitor (SC560) and selective COX2 inhibitor (nimesulide) separately had no significant effect on blastocyst implantation while combination of both inhibitors in lower dose showed partial delay in implantation by more than 24h and became implanted beyond the window of implantation, i.e. on D6 but these implantation sites were significantly reduced on D10 and the pregnancy is lost in significant number. However, the higher doses of inhibitors in combination completely prevented implantation. Embryos retrieved from these treated mice showed significantly lower number of embryonic cells (77+/-3.3 and 65.2+/-3.9) than the optimum number of embryonic cells (93.4+/-2.6). The lower doses of both the inhibitors reduced uterine PGE2 and PGI2 content on D5 but did not inhibit as efficiently as higher doses. In addition, our immunohistochemistry result shows that there was no COX1 and COX2 localization on D5 of treated mice but COX2 begins expressing on D6 like normal D5 of pregnancy. Therefore, we can conclude that embryos implanted after the delay showed defective post-implantation development because of lower number of embryonic cells of implanting blastocyst and implantation beyond the proper time in window of receptivity.     

Collagen concentrations in dissected tissue compartments of rat uterus on days 6, 7 and 8 of pregnancy.

Implantation and non-implantation sites were dissected into myometrial and stromal components; a decidual/embryonic region was obtained on Days 7 and 8 of pregnancy. The concentration of collagen (as a percentage of the dry weight of tissue), measured by hydroxyproline analysis, was significantly lower in the implantation regions than in the non-implant regions in all areas studied. The concentrations in the antimesometrial myometrium and stroma of the implantation region remained the same over the days studied. In contrast, the mesometrial collagen concentration in the implantation region declined from Day 6 to Day 8 of pregnancy. Collagen concentration was low within the decidual/embryonic tissue on Days 7 and 8 of pregnancy. Remodelling of collagen within the embryonic area appears to be an important feature of the uterine response to implantation in rats.     

Embryonic survival at day 9, 21 and 35 of pregnancy in intact and unilaterally oviduct ligated multiparous sows

To investigate the effect of uterine space on timing of embryonic mortality, multiparous sows were left intact (CTR; n=42) or subjected to unilateral oviduct ligation (LIG; n=23), after their first post wean oestrus. Intact sows were killed at day 9 (n=10), day 21 (n=15), or day 35 (n=17), and LIG sows were killed at day 21 (n=11) or day 35 (n=12) of gestation. At day 9, 92% of ovulations were represented by an embryo. At day 21, embryonic mortality was 24% and was not altered by increasing uterine space. At day 35, space per embryo was twice as large in LIG sows (30±3 v. 16±0.8 cm), and implantation length tended to be larger (19.0±1.2 v. 15.5±1.3 cm). Between day 21 and day 35, CTR sows lost another 8% to 14% of their embryos, whereas LIG sows lost none. Embryos tended to be heavier (4.9±0.2 v. 4.3±0.3 g) in LIG sows. In conclusion, embryonic loss in multiparous sows is 24% by day 21 and is not related to space, whereas after day 21 limited space causes additional 8% to 14% embryonic mortality in intact sows only.     

Evidence of increased endometrial vascular permeability at the time of implantation in the short-nosed fruit bat, Cyanopterus sphinx

Early embryonic development and implantation were studied in tropical short-nosed fruit bat Cyanopterus sphinx. We report preimplantation development and embryo implantation. Different stages of cleavage were observed in embryo by direct microscopic examination of fresh embryos after retrieving them either from the oviduct or the uterus at different days, up to the day of implantation. Generally, the embryos enter the uterus at the 8-cell stage. Embryonic development continued without any delay and blastocyst were formed showing attachment to the uterine epithelium at the mesometrial side of the uterus. A distinct blue band was formed in the uterus. The site of blastocyst attachment was visualized as a blue band following intravenous injection of pontamine blue. Implantation occurred 9+/-0.7 days after mating. This study reports that bat embryonic development can be studied like other laboratory animals and that this bat shows blue dye reaction, indicating the site and exact time of implantation. This blue dye reaction can be used to accurately find post-implantational delay. We prove conclusively that this species of tropical bat does not have any type of embryonic diapause.     

Navigating the site for embryo implantation: Biomechanical and molecular regulation of intrauterine embryo distribution

The distribution of intrauterine embryo implantation site(s) in most mammalian species shows remarkably constant patterns: in monotocous species such as humans, an embryo tends to implant in the uterine fundus; in polytocous species such as rodents, embryos implant evenly along the uterine horns. These long-time evolved patterns bear great biological significance because disruption of these patterns can have adverse effects on pregnancies. However, lack of suitable models and in vivo monitoring techniques has impeded the progress in understanding the mechanisms of intrauterine embryo distribution. These obstacles are being overcome by genetically engineered mouse models and newly developed high-resolution ultrasound. It has been revealed that intrauterine embryo distribution involves multiple events including uterine sensing of an embryo, fine-tuned uterine peristaltic movements, time-controlled uterine fluid reabsorption and uterine luminal closure, as well as embryo orientation. Diverse molecular factors, such as steroid hormone signaling, lipid signaling, adrenergic signaling, developmental genes, ion/water channels, and potentially embryonic signaling are actively involved in intrauterine embryo distribution. This review covers the biomechanical and molecular aspects of intrauterine embryo distribution (embryo spacing at the longitudinal axis and embryo orientation at the vertical axis), as well as its pathophysiological roles in human reproductive medicine. Future progress requires multi-disciplinary research efforts that will integrate in vivo animal models, clinical cases, physiologically relevant in vitro models, and biomechanical/computational modeling. Understanding the mechanisms for intrauterine embryo distribution could potentially lead to development of therapeutics for treating related conditions in reproductive medicine.     

Effects of Annona squamosa extract on early pregnancy in rats.

Annona squamosa Linn., family Annonaceae, is said to show varied medicinal effects, including insecticide, antiovulatory and abortifacient. The purpose of present study was to investigate if A. squamosa seed aqueous extract, in doses higher than that popularly used to provoke abortion, interferes with reproductive performance, and to correlate the ingestion of this extract with possible alterations in rat embryonic implantation. Doses of 300 mg/kg (Treated Group I, n = 17) and 600 mg/kg (Treated Group II, n = 12) body wt. were administered by gavage, during days 1 to 5 of pregnancy (preimplantation period). The control group (n = 13) received water in the same manner, during the same period for comparison with experimental groups. The animals were euthanized on day 10 of pregnancy. Treatment of dams during the preimplantation period showed no signs of toxicity, and no alteration in the corpora lutea, implantations and embryo in terms of development numbers. The percentage of preimplantation and postimplantation losses in treated groups I and II did not differ from those of control. Treatment with aqueous extract of A. squamosa seeds caused no morphological change in the endometrium. The absence of morphological alterations in uterine epithelial cells in treated groups I and II permitted a viable embryonic implantation, as verified by the number of embryos in development at day 10 of pregnancy. Thus, A. squamosa seed aqueous extract did not interfere with the reproductive performance of pregnant rats.     

Ultrasonographic and hormonal monitoring of pregnancy in the saddle back tamarin,Saguinus fuscicollis

Ultrasonography was used in six saddle back tamarin females (Saguinus fuscicollis) to diagnose pregnancy, monitor the patterns of uterine growth and embryonic/foetal development and examine the incidence loss of single embryos/foetuses. Pregnancy was reliably diagnosed 17 days after conception, 10 days earlier than by plasma progesterone measurement. The patterns of uterine and embryonic/foetal growth paralleled those reported for the common marmoset, including a delay in embryonic development. The results support the hypothesis of retardation of organogenesis in most callitrichid species. Individual embryos could be reliably identified from day 50 of pregnancy; a loss of single embryos/foetuses after this stage did not occur. All pregnancies were carried to term, resulting in five times twins and one singleton. The smaller litter size compared to the common marmoset may be due to loss of single embryos at earlier stages of pregnancy or to a lower ovulation rate.     

The effect of age on multiple ovulation rates, multiple pregnancy rates and embryonic vesicle diameter in the mare

Numerous and conflicting reports exist regarding factors that may effect mare reproductive performance, in particular multiple ovulation (MO) and its consequences. Sequential ultrasonic examination was used to monitor 3075 ovulations in 1581 mainly Thoroughbred mares to ascertain: whether increasing age is associated with an increase in MO; whether this is counteracted by an increase in embryo mortality (EM) prior to Day 13; and whether this embryonic loss may be associated with small-for-age embryonic vesicles (Days 13/14). Overall ovulation rate was 1.31, MO occurring in 29.3% of cycles. MO incidence significantly (p<0.05) increased with age (20.7% in 2-4-year olds compared to 35.6% in 17-19-year olds). 25.2% of MO were apparent as multiple pregnancies (MP) (40.0% of all pregnancies arising from MO) and 37.8% as single pregnancies (SP) at Days 13/14. Older mares demonstrated significantly (p<0.001) lower pregnancy rates and of those pregnant, significantly (p<0.01) fewer were MP than younger mares. Observation of 1442 embryonic vesicles failed to demonstrate any consistent significant association between age and vesicle size in single ovulating (SO) or MO mares on Days 13/14. We conclude that: (i) increasing age was significantly (p<0.05) associated with increasing incidence of MO; (ii) increasing age was significantly associated with a decreasing incidence of pregnancy/ovulation (p<0.001), and MP (p<0.01), at Days 13/14; (iii) there was no consistent significant association between mare age and vesicle size.     

Postnatal deletion of Wnt7a inhibits uterine gland morphogenesis and compromises adult fertility in mice

The success of postnatal uterine morphogenesis dictates, in part, the embryotrophic potential and functional capacity of the adult uterus. The definitive role of Wnt7a in postnatal uterine development and adult function requires a conditional knockout, because global deletion disrupts müllerian duct patterning, specification, and cell fate in the fetus. The Wnt7a-null uterus appears to be posteriorized because of developmental defects in the embryo, as evidenced by the stratified luminal epithelium that is normally found in the vagina and the presence of short and uncoiled oviducts. To understand the biological role of WNT7A after birth and allow tissue-selective deletion of Wnt7a, we generated loxP-flanked exon 2 mice and conditionally deleted Wnt7a after birth in the uterus by crossing them with Pgr(Cre) mice. Morphological examination revealed no obvious differences in the vagina, cervix, oviduct, or ovary. The uteri of Wnt7a mutant mice contained no endometrial glands, whereas all other uterine cell types appeared to be normal. Postnatal differentiation of endometrial glands was observed in control mice, but not in mutant mice, between Postnatal Days 3 and 12. Expression of morphoregulatory genes, particularly Foxa2, Hoxa10, Hoxa11, Msx1, and Wnt16, was disrupted in the Wnt7a mutant uteri. Conditional Wnt7a mutant mice were not fertile. Although embryos were present in uteri of mutant mice on Day 3.5 of pregnancy, blastocyst implantation was not observed on Day 5.5. Furthermore, expression of several genes (Foxa2, Lif, Msx1, and Wnt16) was reduced or absent in adult Wnt7a-deleted uteri on Day 3.5 postmating. These results indicate that WNT7A plays a critical role in postnatal uterine gland morphogenesis and function, which are important for blastocyst implantation and fertility in the adult uterus.     

Mucin and proteoglycan functions in embryo implantation

Embryo implantation is a complex series of events that involves changes in pattern of expression of embryonic as well as uterine cell surface components. In the case of the embryo, these changes are driven by the developmental program. In the case of the uterus, these changes are triggered by both maternal hormonal influences as well as embryo-derived factors. Aspects of the implantation process vary among species; however, interaction between the external surface of the embryonic trophectoderm and the apical surface of the lumenal uterine epithelium is a common event. Progress is being made in defining the molecular players in these cell surface interactions. Large-molecular-weight mucin glycoproteins such as MUC1 are present at the apical surface of the uterine epithelium under most conditions. Under most circumstances, these mucins appear to protect the mucosal surface from infection and the action of degradative enzymes. These mucins are antiadhesive and also appear to represent a barrier to embryo attachment. Consistent with this model, reduction of mucin expression is observed in uterine lumenal epithelia in many species. Nonetheless, mucin expression persists in the human uterus during the proposed receptive phase. It is possible that mucin loss is localized to implantation sites in humans. Alternatively, mucins may function differently within the context of human implantation than in other species. Studies primarily performed in mice indicate that heparan sulfate proteoglycans, in particular, perlecan, appears on the exterior trophectodermal surface coincident with the acquisition of attachment competence. Various in vitro studies indicate that heparan sulfate proteoglycans support embryo attachment activity that may represent an early event in embryo–uterine interaction. Uterine epithelia cells express several complementary heparan sulfate-binding proteins that may participate in these attachment processes. Use of molecular genetic approaches in mouse models, as well as careful studies of the expression and function of these molecules in the context of implantation in various species are beginning to shed light on the key molecular events of implantation. BioEssays 20 :577–583, 1998. © 1998 John Wiley & Sons Inc.     

Expression Pattern of E- and P-Cadherin in Mouse Embryos and Uteri during the Periimplantation Period

Periimplantation mouse embryos and uterine tissues were examined by means of immunohistochemistry for their expression of the Ca²⁺ dependent cell-cell adhesion molecules, E- and P-cadherin. E-cadherin was detected in all embryonic cells during periimplantation stages, and also detected in the uterine epithelium. When blastocysts attached to the uterine epithelium, E-cadherin was detected at implantation sites between the mural trophectoderm and the uterine epithelium on 5 day of pregnancy. P-cadherin was first detected in the mural trophectoderm on 4.5-day blastocysts, and then detected in the ectoplacental cone, giant cells and visceral endoderm from 5.5 day.
P-cadherin was also detected in the maternal uterine decidual cells from 5.5 day. After degeneration of uterine epithelial cells, giant cells make direct contact with uterine decidual cells, and P-cadherin was detected at contact sites between these cells.
Thus, the complicated process of implantation seems to be supported by temporal and spatial expression of the multiple classes of cadherins.     

Development of pig blastocysts in a uterine environment advanced by exogenous oestrogen

Routine embryo transfer techniques were used to establish recipient groups in which blastocysts were either asynchronous (blastocysts 24 h behind recipient uterus) or synchronous with their uterine environment. Oestradiol valerate (5 mg) was administered on Day 11 of the recipient's cycle to stimulate release of uterine secretion in the synchronous gilts (Group SE) and one group (AE) of asynchronous gilts. The gilts in the other asynchronous group (Group AC) were injected with vehicle (sesame oil). Embryos recovered on Day 14 by hysterectomy and flushing were evaluated for morphological development. Oestradiol treatment resulted in a failure of blastocyst development in Group AE gilts only. Recoverable oestradiol in the uterine flushings was increased in gilts in Groups AC and SE which contained elongated blastocysts. Plasmin inhibitor levels were lower in Groups AC and SE while PGF tended to be increased. Acid phosphatase activity was higher and recoverable Ca2+ was lower in Groups AE and SE. Failure of blastocyst development in Group AE is believed to have resulted from a failure to undergo trophoblastic elongation due to premature alteration of the uterine environment at a critical period of blastocyst development or from the presence of an unfavourable uterine environment for blastocyst attachment and development shortly after Day 12.     

Spatiotemporal expression of cyclooxygenase 1 and cyclooxygenase 2 during delayed implantation and the periimplantation period in the Western spotted skunk

Embryonic development in the western spotted skunk is arrested after blastocyst formation for about 200 days. This developmental arrest is believed to be due to insufficiency of uterine conditions to support continuous development. Implantation and decidualization are defective in cyclooxygenase 2 (Cox2)-, but not Cox1-, deficient mice. We therefore used Northern and in situ hybridization to investigate changes in uterine expression of Cox1 and Cox2 genes during various stages of pregnancy in the spotted skunk. Cox1 was constitutively expressed at all stages of pregnancy examined, but it did exhibit localized up-regulation in the trophoblast and necks of uterine glands at early implantation sites. Cox2 expression was highly regulated with little or no expression during delayed implantation. Cox2 expression was first detected in the uterus and trophoblast prior to blastocyst attachment and remained detectable for 5-6 days after blastocyst attachment. Cox2 expression was also localized in the luminal and glandular epithelia of uterine segments located between implantation chambers. Changes in Cox expression were not correlated with the abrupt increase in uterine weight that occurs simultaneously with renewed embryonic development but was correlated with an influx of serum proteins into the uterus observed in a previous study.