In mammals, pregnancy is an irreversible and complicated event. The mammalian uterus requires many physiological and morphological changes for pregnancy-associated events including implantation, decidualization, placentation and parturition. The failure to complete any events results in implantation failure, spontaneous miscarriage or abnormal parturition, including preterm birth. These events are primarily regulated by ovarian estrogen and progesterone (P4). P4 and estrogen are produced in the ovary throughout pregnancy in mice, but in humans, hormonal support switches from the ovary to the placenta. The first direct interaction between embryo and uterus is implantation. In humans, about 75% of unsuccessful pregnancies are believed to result from defective implantation. Therefore, a better understanding of the molecular mechanisms associated with implantation would be helpful for the further improvement of clinical treatments. Recent studies using genetically modified mice have given us considerable insight into the molecular mechanisms underlying embryo implantation. In this review, we discuss in the understanding recent advances of the molecular events during implantation, especially focusing on the roles of estrogen and P4 signaling. We also offer our thoughts on the as yet unelucidated processes in implantation to guide and stimulate further research in this area.

Synchronization of embryonic development and differentiation of specific uterine cell types to a receptive state is essential for a successful pregnancy. The period of uterine receptivity for implantation is limited. Increased vascular permeability and angiogenesis are hallmarks of the implantation process. Although implantation involves the interaction of numerous signaling molecules, the hierarchical mechanisms that coordinate the embryo—uterine dialog remain poorly understood. This review highlights our knowledge about angiogenesis, uterine receptivity, and hormonal regulation for blastocyst implantation in the mouse. A better understanding of uterine biology during the peri-implantation period would facilitate the further development of reproductive technology.

Pregnancy comprises multiple stages with complex interactions of molecules and cells, and previous studies have clarified that progesterone (P₄) is a key player in pregnancy. Several animal experimental models have been established to address the detailed mechanisms of P₄, and genetically engineered mouse models have especially helped us understand its function. P₄ receptor (PR)-null female mice show no ovulation, while PR co-chaperone FKBP52-null mice exhibit implantation failure with normal ovulation. Moderate supplementation of P₄ rescues implantation failure in FKBP52-deficient mice but does not restore the capability for pregnancy up to full term, resulting in embryo resorption. Supplementation of a large amount of P₄, however, can rescue pregnancy and provide normal reproductive outcomes until parturition. Mouse studies by our groups, and others, have also shown that epigenetic regulation of uterine P₄-PR signaling, P₄-induced molecular crosstalk between the epithelium and stroma and uterine proliferation-differentiation switching are indispensable for successful implantation. Collectively, P₄ orchestrates the whole process of pregnancy in spatiotemporal manners, eventually integrating them toward successful parturition. In this review article, we review the literature on the uterine functions of P₄ in pregnancy, with a special focus on the knowledge gained about embryo implantation by studies utilizing mouse models.

During the menstrual cycle, the human endometrium undergoes dramatic changes, including cyclical proliferation, differentiation and menstruation, under the stringent control of ovarian steroid hormones. Endometrial stromal cells (ESCs) spontaneously differentiate into decidual cells in response to increased progesterone and intracellular cyclic adenosine monophosphate (cAMP) levels during the mid-secretory phase of the cycle. This process, termed decidualization, is strictly defined as the morphological and biochemical reprograming of the ESCs and is required for successful pregnancy. In pregnancy, the decidua functions as a critical barrier between the mother and fetus by modulating trophoblast invasion and the immune response. We recently demonstrated the involvement of a novel cAMP target, an exchange protein directly activated by cAMP (EPAC), in the process of decidualization. This review presents the molecular mechanisms of decidualization regulated by progesterone and the cAMP signaling pathway and highlights the role of EPAC in the process of decidualization.

The morphological characterization of ovaries and ovarian follicular oocytes obtained from the owl monkey is detailed in the present paper. In vitro maturation of oocytes, using a method which has proved successful for other mammalian oocytes, was also evaluated and the maturation rate was compared with that obtained with squirrel monkey oocytes. The ovaries of the owl monkey are oval in shape (long axis of about 10 mm, short axis about 7 mm) and their oocytes are spherical. The mean diameter of owl monkey oocytes is significantly larger than that of squirrel monkey oocytes (145.7 ± 16.3 µm vs 112.0 ± 12.4 µm). The owl monkey oocytes were incubated in HTF medium containing 10% FBS at 37 °C (5% CO₂ in air) for 22–23 hours. The in vitro maturation rate of the owl monkey oocytes was higher than that of the squirrel monkey oocytes (83.3% vs 40.0%); therefore, our maturation conditions were as suitable for them. This study is the first to detail differences in the follicular oocytes of the owl monkey and squirrel monkey. Further studies using owl monkey gametes might yield results leading to a greater understanding of primate reproduction.

Frozen-thawed embryo transfers, whose number has risen considerably in recent years, reportedly result in heavier birth weights than fresh embryo transfers. To find out what this difference means and the stage at which it becomes manifest during fetal development, we studied birth weight and gestational sac size, which reflects development immediately following implantation, in 365 single pregnancies employing fresh embryo transfer and 227 employing frozen-thawed embryo transfer. Comparison of fresh embryo transfers and frozen-thawed embryo transfers revealed that average birth weights were significantly higher in the latter, with average values ± SD of 2896.0 ± 515.7 g and 3060.0 ± 529.2 g, respectively. Transvaginal ultrasound showed significantly larger average gestational sac diameters at 21, 22, 23, 28, 29 and 30 days after fertilization in frozen-thawed embryo transfers. We speculate these results are explained mainly by hormone replacement therapy in frozen-thawed embryo transfer cycles exerting a more positive influence on the endometrium, promoting smoother implantation, greater development during early pregnancy, and significant increases in birth weight. Amidst concerns regarding the impact exerted on fetuses by the artificial operations entailed by in vitro fertilization and embryo transfers, these findings may serve as evidence of the safety of frozen-thawed embryo transfers.

At our clinic, a successfully fertilized human oocyte is defined as one with two polar bodies and two pronuclei (PN) 18 h after ICSI, but when some oocytes do not form PN. Recently, a successful pregnancy and live birth following artificial oocyte activation (AOA; using Ca² ionophore) of ICSI-treated one-day-old unfertilized oocytes was reported. In this study, we performed AOA on PN-lacking unfertilized oocytes, 18 h after ICSI (defined as Day 1-AOA, since ICSI=Day 0). The abilities of these oocytes to form PN and develop were compared with those of oocytes fertilized by ICSI (defined as No-AOA). Piezo-ICSI was performed on 168 oocytes, 50 of which failed to fertilize (0PN) but were subsequently artificially activated. 2PN formation rate was significantly lower in Day 1-AOA, and the rates of 1PN and ≥4PN were significantly higher than in No-AOA. In Day 1-AOA, 8 oocytes formed 2PN, 6 zygotes underwent cleavage and 2 of them developed to the morula stage, but none formed blastocysts. The rates of cleavage, morula, and blastocyst formation were significantly lower in Day 1-AOA than in No-AOA. These results suggest that the AOA protocol has room for improvement in the activation of unfertilized oocytes 18 h after ICSI.

The objective of this study was to develop a transportation system for fresh porcine embryos in a chemically defined medium without reducing their viability using a novel embryo carrier. When embryos were introduced in 0.25-ml straws with porcine blastocyst medium (PBM) and given a vibration load on a shaker for 20 h to mimic transportation conditions at 38 °C, there was no significant difference in survival rates between the vibration and no-vibration groups. Next, an embryo carrier was developed that can maintain interior conditions of 5% CO₂ and 38 °C for the transportation of embryos. The embryos on Day 5 after insemination were divided into three groups. In the first group, the embryos were introduced to straws with PBM and the straws were sealed (sealed group). In the second group, the embryos were transported in a tube with a CO₂ pouch (CO₂ gas group). They were transported by a door-to-door delivery service. In the last group, the embryos were cultured in an incubator (not-transported group). Although the survival rate of embryos in the sealed group was significantly lower than that of not-transported group, the survival rates and the hatching rate of the embryos in the CO₂ gas group were similar to those of not-transported group of embryos (100% vs 97.4% and 57.9% vs 47.5%, respectively). Our results demonstrate that in vivo-derived porcine embryos can be transported without harm in PBM using our carrier.