Early implantation stages in the marmoset monkey (Callithrix jacchus)

The morphology of the initial stages of implantation in the marmoset monkey (Callithrix jacchus) was studied by obtaining embryos and associated endometrium at timed intervals after ovulation. Estrus cycles were detected by measuring daily levels of plasma progesterone. Following a short follicular phase, circulating levels of progesterone above 20 ng/ml were taken as representing day 1 after ovulation. On this basis, single, twin, and triplet embryos were recovered from six perfused-fixed females on days 13, 16, 19, 23, and 29 after ovulation and prepared in resin for light microscopy. Early implantation stages, 13 and 16 days after ovulation, were characterized by the intrusion of syncytial trophoblast between epithelial cells of the endometrium with minimal cellular damage. Some hyperplasia of epithelium at the margin of the implantation site was evident. The consolidation of the initial attachment was achieved by an increase in syncytial trophoblast underlying the inner cell mass of the embryo which rapidly surrounded and breached maternal capillaries. Although initially separate, the chorions of twin or triplet embryos started to fuse by day 19 after ovulation. This process was complete by day 29 such that embryos shared a common uterine exocoelom surrounded by continuous trophoblast. It was concluded that implantation in the marmoset monkey commenced on days 11-12.5 after ovulation and involved an intrusive mechanism. Although trophoblast penetration of endometrium was superficial, maternal capillaries were tapped at an early stage of implantation. The fusion of chorions of twins and triplets first occurred around day 19 after ovulation.     

Immunohistochemical localization of prostaglandin H synthase in the embryo and uterus of the mouse from ovulation through implantation

Prostaglandins (PGs) in the embryo and endometrium are involved in processes that are important for implantation. Although the presence of PGs (PGE2, PGF2 alpha, PGI2) in decidualized endometrium has been widely reported, less is known about the capacity of the pre-implantation embryo to synthesize PGs. Prostaglandin H (PGH) synthase is necessary for the production of PGs. Using an immunohistochemical method, PGH synthase was localized in the mouse embryo and uterus from superovulation through embryo implantation. No PGH synthase was detected in oocytes at the time of ovulation or in single-cell embryos 1 day post-fertilization (PF). Circular areas of immunostaining became evident in the cytoplasm of blastomeres at the morula stage (day 3 PF). After implantation (day 5 PF), a low level of PGH synthase reactivity was observed in embryonic cells; no PGH synthase was detected in the embryo by day 7 PF. The endometrial glands exhibited maximal immunostaining by day 3 PF, and after implantation, PGH synthase appeared in decidual cells along the border of placentation. Low levels of PGH synthase reactivity were detected in myometrial cells during the period after superovulation through day 7 PF. This is the first demonstration of PGH synthase in the mouse embryo prior to apposition with glandular endometrial epithelium, supporting the hypothesis that the embryo has the potential to produce PGs that may mediate autocrine and/or paracrine responses at the time of nidation.     

TGF superfamily and MMP2, MMP9, TIMP1 genes expression in the endometrium of women with impaired reproduction

During the putative "implantation window", a period of maximal endometrial receptivity that spans 7-9 days after ovulation, a series of changes on the structural and molecular level occur that render the endometrium susceptible to implantation for the human embryo. Many members of the TGFbetas are expressed by human endometrium at different stages of menstrual cycle. Also studies regarding the MMP2 gene expression and activity of MMP2 in the implantation window have shown a higher expression and activity of MMP2 in women with impaired fertility. We have examined by RT-PCR the expression of TGFbeta2 and MMP2, MMP9 and TIMP1 in 28 patients with idiopathic infertility, 16 patients with unexplained recurrent miscarriage and 16 control women were enrolled in this study. Seven to nine days after ovulation endometrial biopsy by Pipelle or hysteroscopy was performed to assess the expression of TGFbeta2 , MMP2, MMP9 and TIMP1. We found that in endometria from women with idiopathic infertility TGFbeta2 expression was 2.8 fold higher than in endometria from control group and 2.1 fold higher in endometrial samples from women with unexplained recurrent miscarriage compared to the control group. The MMP2, MMP9 and TIMP1 expression in endometrial samples revealed no significant differences between the study groups and control group. There was a statistically significant negative correlation between TGFbeta2 and MMP9 expression in endometria from women in control group. The present investigations suggest that dysregulated TGFbeta2, MMP2, MMP9 and TIMP1 expression are associated with infertility and early pregnancy loss. However the exact mechanism of how overexpression of endometrial TGFbetaand MMPs interferes with implantation may be more complex.     

Analysis of intra-uterine fluid motion induced by uterine contractions

Evaluation of the fluid flow pattern in a non-pregnant uterus is important for understanding embryo transport in the uterus. Fertilization occurs in the fallopian tube and the embryo (fertilized ovum) enters the uterine cavity within 3 days of ovulation. In the uterus, the embryo is conveyed by the uterine fluid for another 3 to 4 days to a successful implantation site at the upper part of the uterus. Fluid movements within the uterus may be induced by several mechanisms, but they seem to be dominated by myometrial contractions. Intra-uterine fluid transport in a sagittal cross-section of the uterus was simulated by a model of wall-induced fluid motion within a two-dimensional channel. The time-dependent fluid pattern was studied by employing the lubrication theory. A comprehensive analysis of peristaltic transport resulting from symmetric and asymmetric contractions is presented for various displacement waves on the channel walls. The results provide information on the flow field and possible trajectories by which an embryo may be transported before implantation at the uterine wall.     

Timing of multiple ovulations in the ewe after treatment with FSH or PMSG with and without GnRH

Ovulation rate, median time to first ovulation, median time of all ovulations and median time from first to last ovulation were studied by repeated laparoscopy in Merino ewes. Treatments with FSH or PMSG significantly affected ovulation rate (8.4 +/- 0.81 and 7.3 +/- 1.21 respectively, P less than 0.05) and in median time of all ovulations (60 and 54 h respectively after progestagen sponge removal, P less than 0.05). Differences in the median time to first ovulation (60 and 48 h) and median time from first to last ovulation (6 and 6 h) for the respective treatments were not significant. The synchrony of ovulation after both treatments was adversely affected by (1) the occurrence of premature ovulations before the onset of superovulation, (2) variability in the time of commencement of superovulation, and (3) variability in the time from first to last ovulation. Administration of GnRH synchronized the timing of ovulation with both gonadotrophin treatments. This synchrony was due to a reduction in the period during which superovulation began and in the interval from first to last ovulation. The median time of all ovulations was significantly less with FSH + GnRH than with PMSG + GnRH (45 and 48 h after progestagen sponge removal, respectively, P less than 0.05). Administration of GnRH at 16, 20 or 24 h after progestagen sponge removal significantly affected all traits examined except ovulation rate. Administration at 20 and 24 h produced an equally good synchrony of ovulation which was better than that obtained at 16 h. We suggest that the use of GnRH in embryo collection programmes appears justified and is likely to improve embryo yields due to improved rates of fertilization.     

Ovulation, fertilization and early embryonic development in the bitch (Canis familiaris)

Using circulating plasma hormone estimations, ovulation was monitored in bitches. The results obtained indicate that the timing of ovulation bears little relationship to alterations in sexual behaviour. The bitches were killed and reproductive tracts were removed at various intervals after ovulation and ova or embryos were recovered. The embryo stages were assessed visually and some were investigated histologically. Embryonic development, to early blastocyst stage, took place within the oviducts during the first 12 days after ovulation and there was a marked increase in size between the early and late blastocyst. A culture system using cells from the uterine tube supported the development of one 1-cell embryo to the morula stage.     

Transformation of endometrium and fertility in late stages of pseudopregnancy in the rabbit

Ovulation was induced in rabbits between Days 14 and 18 of pseudopregnancy by an intravenous injection of hCG. Induction of ovulation from Day 16 onwards led to normal progestational endometrial transformation. In rabbits injected on Day 14 or 15, a normal preimplantation endometrial morphology developed, but not earlier than 7 days after hCG (Day 14/15 + 7). Uteroglobin secretion was advanced during the second pseudopregnancy. After mating or artificial insemination, fertility was greatest on Day 18 of pseudopregnancy. Conception failed on Day 14 and embryo transfers were unsuccessful on Day 14 + 1. Transfers performed on Day 14 + 3, however, led to implantation and offspring, even though endometrial morphology did not correspond to the normal Day 3 preimplantational morphology at the time of transfer. We conclude that endometrial transformation typical of normal pseudopregnancy can be induced by ovulation during the regeneration phase of pseudopregnancy from Day 16 onwards; fertilization and implantation can be achieved as early as Day 15 of pseudopregnancy; an oestrous period with high mating activity and fertility occurs about 3 days later; and Day 14 after hCG represents a limited time of functional change from pseudopregnancy to a fertile uterine cycle in the rabbit.     

Measurement of early pregnancy factor activity for monitoring the viability of the equine embryo

The viability of embryos before flushing from donor mares (n = 5) and after transfer to recipient mares (n = 7) was monitored in mare serum by detecting early pregnancy factor (EPF) using the rosette inhibition test (RIT). The EPF activity was measured in donor mares before and after natural mating at natural estrus; after ovulation on Days 2, 5 and 8; and after embryo flushing (Day 8) on Days 8, 9, 10 and 13 after ovulation. The collected embryos were transferred immediately after flushing. The EPF activity in recipient mares were measured on the day of transfer and after embryo transfer on Days 1, 2, 3 and 5. Pregnancy was confirmed on Day 12 to 14 after embryo transfer. The mean EPF activity of donor mares was increased to the pregnant level (> an RI titer score of 10) on Day 2 after ovulation. Two days after flushing the embryos, the EPF activity of donor mares had decreased to the nonpregnant level. Among the 7 recipient mares, 3 mares were diagnosed pregnant on Day 12 after embryo transfer with ultrasound. The EPF activity of the pregnant recipient mares was increased above the minimum level observed in pregnant mares on Days 2 to 3 after transfer. However, among the nonpregnant recipient mares after embryo transfer, the EPF activity of 3 mares remained at the pregnant level only 2 to 3 d and then declined to the nonpregnant level. In one recipient mare, EPF activity did not reach the pregnant level throughout the sample collection. The results of this study indicated that equine EPF can be detected in serum of pregnant mares as early as Day 2 after ovulation. From our observation, we conclude that the measurement of EPF activity is useful for monitoring the in vivo viability of equine embryos and early detection of embryonic death.     

The effect of clomiphene citrate on blastocyst development and implantation in the rabbit

Summary The induction of ovulation with clomiphene citrate (CC) in human patients results in a high ovulation rate but achieves a relatively low pregnancy rate. To clarify the possible role of CC in interfering with the normal reproductive physiology and embryology, we have used our rabbit model and transferred 4-day-old blastocysts from untreated donors to CC-treated pseudopregnant recipients and from CC-treated donors to untreated pseudopregnant recipients to study embryonic development and implantation. Each group was further subdivided into two subgroups, one receiving CC before and the other after ovulation. CC was administered subcutaneously in three consecutive doses of 10 mg/kg body weight. Ovulation was induced with pregnant mare serum gonadotropin (PMS) and human chorionic gonadotropin (hCG).The implantation rate of the control group, evaluated on day 8 of pregnancy, reached 62.0%. When recipients were treated with CC before ovulation, implantation rate was reduced to 18.8% (P < 0.0002), and to 20.0% (P < 0.003) when CC was administered after ovulation. The implantation rate of blastocysts transferred from donors, treated before ovulation, is 22.2% (P < 0.0055), however, reached 70.8% when treatment was started after ovulation. All implantations were analysed microscopically and showed normal morphological features.Our results demonstrate a potential multiple effect of CC, first on the endometrium by altering its receptivity for the implanting conceptus, second, on tubal physiology by altering egg transport, and finally on ovum maturation before ovulation interfering with development of blastocysts. These parameters may all result in rapid decrease in establishment of implantations and in turn in very low pregnancy rates.     

Ovulation rate, embryo mortality and intrauterine growth retardation in obese swine with gene polymorphisms for leptin and melanocortin receptors

The Mediterranean Iberian pigs are obese genotypes, due to a leptin resistance syndrome related to leptin receptor gene polymorphisms. The Iberian pig is affected by a lower prolificacy when compared to lean breeds, and thus may constitute a good animal model for adverse effects of obesity in reproductive performance. The present study determined possible differences in rates of ovulation and embryo implantation and later incidence of embryo mortality and intrauterine growth retardation (IUGR) in sows of Iberian breed (n = 23) and highly-prolific lean commercial crosses (Large White × Landrace, n = 17) at two critical periods of swine pregnancy: Day 21 (just after achievement of trophoblast attachment) and Day 35 (just after completion of the transition from late embryo to early foetal stage). Two different reproductive performances were identified in the Iberian breed; 58.3% of the females had lower ovulation rates than LWxL (13.2 ± 2.3 vs 22.5 ± 1.6, P < 0.05), but 41.7% had a similar number of ovulations (18.2 ± 3.9). However, those Iberian sows having high ovulatory rates showed a high incidence of regression of corpora lutea and embryo losses between Days 21 and 35, which was not found in Iberian females with low ovulation rates; therefore, the number of viable embryos was similar in both Iberian groups (8.2 ± 1.0 and 8.4 ± 1.0) and lower that in highly-prolific LWxL (14.8 ± 1.8, P < 0.05). At Day 35, a total of 167 conceptuses (73 LWxL and 94 IB) were evaluated for IUGR. The LWxL conceptuses were longer and wider than Iberian (69.5 ± 0.1 and 64.4 ± 0.1 vs 49.9 ± 0.1 and 41.9 ± 0.1 mm, P < 0.0001). However, Iberian conceptuses were heavier than LWxL (107.4 ± 6.6 vs 68.6 ± 2.4 g) due to a lower quantity of fluids and a higher development of the placental tissues in comparison to the embryo itself. In conclusion, current study indicates a bimodal effect of obese genotypes on reproductive performances, either by lowering ovulation rate or by increased embryo losses in the first third of pregnancy.     

Platelet-activating factor: a paracrine factor in preimplantation stages of reproduction?

Platelet-activating factor (PAF) exerts its actions through activation of specific membrane binding sites found in a variety of tissues, including hypothalamus and endometrium. PAF has been identified in several reproductive tissues (i.e. embryo, ovary, uterus, and spermatozoon). In the uterus, PAF levels are hormonally controlled, being elevated by progesterone and prostaglandin E2 (PGE2). Antagonists of PAF interfere with sperm function, ovulation, and implantation. The available evidence suggests that PAF may be an important physiological regulator in reproduction.     

Diversification of cyclooxygenase-2-derived prostaglandins in ovulation and implantation

Previous observations of ovulation and fertilization defects in cyclooxygenase-2 (COX-2)-deficient mice suggested that COX-2-derived ovarian prostaglandins (PGs) participate in these events. However, the specific PG and its mode of action were unknown. Subsequent studies revealed that mice deficient in EP(2), a PGE(2)-receptor subtype, have reduced litter size, apparently resulting from poor ovulation but more dramatically from impaired fertilization. Using a superovulation regimen and in vitro culture system, we demonstrate herein that the ovulatory process, not follicular growth, oocyte maturation, or fertilization, is primarily affected in adult COX-2- or EP(2)-deficient mice. Furthermore, our results show that in vitro-matured and -fertilized eggs are capable of subsequent preimplantation development. However, severely compromised ovulation in adult COX-2- or EP(2)-deficient mice is not manifested in immature (3-wk-old) COX-2- or EP(2)-deficient mice, suggesting that the process of ovulation is more dependent on PGs in adult mice. Although the processes of implantation and decidualization are defective in COX-2(-/-) mice, our present results demonstrate that these events are normal in EP(2)-deficient mice, as determined by embryo transfer and experimentally induced decidualization. Collectively, previous and present results suggest that whereas COX-2-derived PGE(2) is essential for ovulation via activation of EP(2), COX-2-derived prostacyclin is involved in implantation and decidualization via activation of peroxisome proliferator-activated receptor delta.     

低能氮离子注入对决明子的生物效应研究

用低能氮离子注入决明子种胚,种子浸泡液电导率显著增加,发芽势和发芽指数显著降低,幼苗主根长度减少,平均鲜重和活力指数显著降低.直接接受离子注入的种胚,其SOD酶活性随注入剂量的增加呈现出先下降再轻微上升的趋势.而没有直接接受离子注入的胚乳,其SOD酶活随注入剂量的增加呈马鞍形曲线.     

Recovery rate and quality of embryos from mares inseminated after ovulation

The aim of this study was to evaluate the quality of embryos and their recovery rate from mares inseminated at different intervals after ovulation. Finnhorse and warmblood mares were inseminated with fresh semen 8 to 16 h, 16 to 24 h, or 24 to 32 h after ovulation. Control mares were inseminated before ovulation. Sixty-seven embryo flushings were performed between Days 7 and 9 after ovulation/insemination. Thirteen mares were not flushed, but their uteri were scanned for pregnancy on Days 14 to 16. Embryo recovery rates decreased as time from ovulation to insemination increased, although embryo quality remained normal as evaluated by morphological criteria and mitotic index. However, postovulatory insemination in this trial appeared to delay embryo development, since the embryos recovered from mares inseminated after ovulation were appreciably smaller and at an earlier stage of development than control embryos recovered from mares inseminated prior to ovulation. Part of this delay in embryo development in the postovulation group could be due to the time needed for sperm capacitation. In addition, as the time from ovulation to insemination increased, embryo development might have been further delayed by defects in the aging oocyte.     

Embryo survival, uterine fluids and tubal SEM in progesterone-asynchronized rabbits

Survival of embryos exposed to several concentrations of uterine proteins and changes in tubal morphology in rabbits given low preovulatory doses of progesterone (P4) that had previously not affected ovulation or fertilization, but caused severe embryo mortality, were studied. In experiment 1, 332 morulae were cultured for 24 h in a control medium containing < 0.5 to > 3.0 mg x mL(-1) of Day 3 uterine fluid proteins. There was no difference in blastocyst development nor implantation to Day 12 following transfer of the blastocysts to recipients, except fewer implants developed in the BSA control. In experiment 2 the oviducts and uteri of control and P4-treated does were examined by SEM for 8 days following ovulation. Secretory cells in the oviducts and to a lesser extent in the uteri were stimulated by P4 treatment for 3 to 4 days after ovulation. Morphology of ciliated cells was unaffected. The subtle changes did not fully account for P4-induced embryo mortality in vivo.     

Ovulation rate, embryo survival and ovarian sensitivity to gonadotrophins in mice selected for litter size and body weight

Single trait selection of mice for either large body size or large litter size resulted in an increased ovulation rate because of possible enhanced ovarian sensitivity to gonadotrophins. There was no difference in pre-implantation embryonic survival in either of the selected lines when compared to control mice. Selection for body weight did not alter post-implantation embryo survival, but fewer fetuses were lost after implantation in the litter size line compared to the control line. Index selection for large body size and small litter size did not change ovulation rate but increased pre- and post-implantation embryonic mortality. Selection for small body size and large litter size increased ovulation rate and decreased early embryonic death.     

Geminin deletion in mouse oocytes results in impaired embryo development and reduced fertility

Geminin controls proper centrosome duplication, cell division, and differentiation. We investigated the function of geminin in oogenesis, fertilization, and early embryo development by deleting the geminin gene in oocytes from the primordial follicle stage. Oocyte-specific disruption of geminin results in low fertility in mice. Even though there was no evident anomaly of oogenesis, oocyte meiotic maturation, natural ovulation, or fertilization, early embryo development and implantation were impaired. The fertilized eggs derived from mutant mice showed developmental delay, and many were blocked at the late zygote stage. Cdt1 protein was decreased, whereas Chk1 and H2AX phosphorylation was increased, in fertilized eggs after geminin depletion. Our results suggest that disruption of maternal geminin may decrease Cdt1 expression and cause DNA rereplication, which then activates the cell cycle checkpoint and DNA damage repair and thus impairs early embryo development.     

Lipid signaling in embryo implantation

A reciprocal interaction between the implantation-competent blastocyst and the receptive uterus is required for successful implantation. Although various molecular pathways are known to participate in this cross-talk, a comprehensive understanding of the implantation process is still missing. Gene expression studies and genetically engineered mouse models have provided evidence that lipid mediators serve as important signaling molecules in coordinating the series of events during early pregnancy including preimplantation embryo formation and development, implantation and postimplantation growth. This review focuses on the roles of two groups of lipid mediators, prostaglandins (PGs) and endocannabinoids, during early pregnancy. Our laboratory has shown that while PGs generated by the cPLA2-cyclooxygenase (COX) system are essential to ovulation, fertilization, and implantation, endocannabinoids are important for synchronizing preimplantation embryo development with uterine receptivity for implantation. A better understanding of these molecular signaling pathways is hoped to generate new strategies to correct implantation failure and improve pregnancy rates in women.     

Factors affecting pregnancy rates and early embryonic death after equine embryo transfer

In the present study, 638 embryo transfers conducted over 3 yr were retrospectively examined to determine which factors (recipient, embryo and transfer) significantly influenced pregnancy and embryo loss rates and to determine how rates could be improved. On Day 7 or 8 after ovulation, embryos (fresh or cooled/transported) were transferred by surgical or nonsurgical techniques into recipients ovulating from 5 to 9 d before transfer. At 12 and 50 d of gestation (Day 0 = day of ovulation), pregnancy rates were 65.7% (419 of 638) and 55.5% (354 of 638). Pregnancy rates on Day 50 were significantly higher for recipients that had excellent to good uterine tone or were graded as "acceptable" during a pretransfer examination, usually performed 5 d after ovulation, versus recipients that had fair to poor uterine tone or were graded "marginally acceptable." Embryonic factors that significantly affected pregnancy rates were morphology grade, diameter and stage of development. The incidence of early embryonic death was 15.5% (65 of 419) from Days 12 to 50. Embryo loss rates were significantly higher in recipients used 7 or 9 d vs 5 or 6 d after ovulation. Embryos with minor morphological changes (Grade 2) resulted in more (P<0.05) embryo death than embryos with no morphological abnormalities (Grade 1). Between Days 12 and 50, the highest incidence of embryo death occurred during the interval from Days 17 to 25 of gestation. Embryonic vesicles that were imaged with ultrasound during the first pregnancy exam (5 d after transfer) resulted in significantly fewer embryonic deaths than vesicles not imaged until subsequent exams. In the present study, embryo morphology was predictive of the potential for an embryo to result in a viable pregnancy. Delayed development of the embryo upon collection from the donor or delayed development of the embryonic vesicle within the recipient's uterus was associated with a higher incidence of pregnancy failure. Recipient selection (age, day after ovulation, quality on Day 5) significantly affected pregnancy and embryo loss rates.     

An assessment of reproductive wastage in sheep

Reproductive wastage was evaluated by relating ovulation rate to lambs born or raised in range finewool ewes over a ten-year period. The results indicate that reproductive losses are of large magnitude and that these losses are concentrated in the period of ovulation to implantation and in death losses of lambs born. In this study, 33.5% of the potential lamb crop was lost in the early period. Multiple ovulating ewes showed the greatest embryonic loss. The data do not deviate from the binomial distribution, thus suggesting that these losses are largely due to chance. Further work is indicated in the first seventeen days after ovulation to examine losses due to fertilization failure, chromosomal abnormalities, and implantation failure. Abortion or absorption of the fetus was indicated in only 4% of the ewes sampled. An average of 16% of the potential lamb crop was lost from birth to weaning, with 73% of these losses occurring in the first few weeks. Management practices need to be developed to reduce these losses, and these would be expected to vary from one producer to the next depending on conditions. Increasing ovulation rate is one possible method of improving reproductive efficiency, as results of this study indicate that for each unit increase in ovulation rate, there is an increase of 0.52, 0.46, 0.33, and 0.23 in the average number of embryos surviving, lambs born, lambs marked and lambs weaned, respectively.