Intraperitoneal administration of mouse kisspeptin-10 to mice during estrus stage induces pseudopregnancy.

A simple method for producing pseudopregnant mice supports pup production. In this study, pregnant ICR were obtained mice without mating with vasectomized mice via administration of mouse Kisspeptin-10 (mKp-10) and transferring blastocysts to the uterus. Blastocyst transfer after mKp-10 administration to mice with gapping and reddish pink vagina resulted in 65.2% (15/23) pregnancies, and 39.1% (34/87) of the transferred blastocysts showed full-term growth. Vaginal smears were observed for accurate estrus cycle determination, and subsequent administration of mKp10 to mice during the estrus stage and blastocyst transfer resulted in 95.2% (20/21) pregnancies and 50.7% (104/205) birth rates. Regarding 2-cell transfer after administration of mKp-10, 100% (8/8) of the mice became pregnant, and 45.0% (36/80) of the embryos were born. Administration of mKp-10 to mice during the estrus stage is a convenient way to generate pseudopregnant mice.

Inducing Pseudopregnancy in Female Mice Without the Need for Vasectomized Males Prior to Non-Surgical Embryo Transfer or Artificial Insemination.

For successfully maintaining pregnancy with embryo transfer or artificial insemination, female recipient mice must be induced into a pseudopregnant state. Female mice are traditionally paired overnight with vasectomized males, and the following morning, the presence of a copulation plug is assessed. To increase the efficiency of producing pseudopregnant females, a cervical manipulation technique has been standardized to be used in combination with non-surgical embryo transfer or artificial insemination techniques in mice. The blunt end of a small plastic rod is inserted vaginally to contact the cervix and is vibrated for 30 s by contact with a trimmer. The procedure is quick and does not require anesthesia or analgesia. This technique increases the reliability and predictability of producing pseudopregnant females and entirely eliminates the requirement for vasectomized males. For CD1 mice, the efficiency of pseudopregnancy induction using cervical manipulation was 83% for females in estrus (N = 76) but only 38% of females in estrus were plugged by vasectomized males (N = 24). Artificial insemination in CD1 mice was performed by estrus synchronization with hormones, cervical manipulation, and the uterine transfer of sperm. Artificial insemination recipients receiving cervical manipulation (N = 76) had a pregnancy rate of 72% and an average litter size of 8.3 pups. This method can also be used to produce pseudopregnant females for non-surgical embryo transfer. Therefore, inducing pseudopregnancy by cervical manipulation is a convenient and efficient alternative to mating with a vasectomized male when performing non-surgical assisted reproduction techniques. Using cervical manipulation provides 3Rs (replacement, reduction, and refinement) benefits for assisted reproduction techniques by reducing the number of animals required and eliminating the necessity for surgically altered males.

Elevated estradiol during a hormone simulated pseudopregnancy decreases sleep and increases hypothalamic activation in female Syrian hamsters.

Sleep disruptions are a common occurrence during the peripartum period. While physical and environmental factors associated with pregnancy and newborn care account for some sleep disruptions, there is evidence that peripartum fluctuations in estrogens may independently impact sleep. However, the impact of these large fluctuations in estrogens on peripartum sleep is unclear because it is difficult to tease apart the effects of estrogens on sleep from effects associated with the growth and development of the fetus or parental care. We therefore used a hormone-simulated pseudopregnancy (HSP) in female Syrian hamsters to test the hypothesis that pregnancy-like increases in estradiol decrease sleep in the absence of other factors. Adult female Syrian hamsters were ovariectomized and given daily hormone injections that simulate estradiol levels during early pregnancy, late pregnancy, and the postpartum period. Home cage video recordings were captured at seven timepoints and videos were analyzed for actigraphy. During "late pregnancy," total sleep time and sleep efficiency were decreased in hormone-treated animals during the white light period compared to pretest levels. Likewise, during "late pregnancy," locomotion was increased in the white light period for hormone-treated animals compared to pretest levels. These changes continued into the "postpartum period" for animals who continued to receive estradiol treatment, but not for animals who were withdrawn from estradiol. At the conclusion of the experiment, animals were euthanized and cFos expression was quantified in the ventral lateral preoptic area (VLPO) and lateral hypothalamus (LH). Animals who continued to receive high levels of estradiol during the "postpartum" period had significantly more cFos in the VLPO and LH than animals who were withdrawn from hormones or vehicle controls. Together, these data suggest that increased levels of estradiol during pregnancy are associated with sleep suppression, which may be mediated by increased activation of hypothalamic nuclei.

Successful induction of pseudopregnancy using sonic vibration in mice.

Embryo transfer (ET) is an essential reproductive technology for the production of new animal strains and maintenance of genetic resources. We developed a method, named Easy-ET, to induce pseudopregnancy in female rats by artificial stimulation using sonic vibration instead of mating with vasectomized males. This study examined the application of this method for the induction of pseudopregnancy in mice. Offspring were obtained from two-cell embryos transferred into females with pseudopregnancy induced using sonic vibration in proestrus on the day before embryo transfer. Furthermore, high developmental rates of offspring were observed when pronuclear and two-cell embryos were transferred to females in estrus that were stimulated on the day of embryo transfer. Genome-edited mice were also obtained using frozen-warmed pronuclear embryos with clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated system (Cas) nucleases introduced using the technique for animal knockout system by electroporation (TAKE) method, which were transferred to females with pseudopregnancy induced on the day of embryo transfer. This study demonstrated that induction of pseudopregnancy by sonic vibration was also possible in mice.

Effect of pseudopregnancy duration in nonpregnant sows on induced lactation.

This study was conducted to examine the effects of different pseudopregnancy periods in nonpregnant sows on artificial lactation induction efficiency and milk composition. Sixteen pseudopregnant sows (n = 4 per group) were treated with 5 mg of estradiol dipropionate at 28 (Group D38), 35 (Group D45), 42 (Group D52), and 49 (Group D59) days after the end of estrus, followed by prostaglandin F2α as 0.175-mg cloprostenol twice at 12 h intervals 10 days later. The overall success rate of lactation induction was 81.3%. The lactation rates were significantly higher in Groups D38, D45, and D59 (100.0%) than in Group D52 (25.0%). The milk immunoglobulin (Ig) G concentration was significantly higher in Group D38 than in Group D59. However, IgA levels and milk compositions (protein, ash, and lactose) did not differ among the groups. Lactation induction was successful between 38 and 59 days of pseudopregnancy. Apart from IgG, pseudopregnancy length did not affect milk components from 38 to 59 days of pseudopregnancy.

Anti-Müllerian hormone concentrations in female rabbits and its relation to spay status, pseudopregnancy and ovarian follicle numbers.

Anti-Müllerian hormone (AMH), known for its role during foetal sexual differentiation, is secreted by the Sertoli cells in males and the granulosa cells in females during post-natal life. As serum AMH concentrations correlate with follicle numbers, AMH is utilized as a marker of ovarian reserve in many species. In dogs and cats, AMH is used as a diagnostic tool to determine spay or neuter status. In the available literature, no research regarding serum AMH levels in rabbits has been published yet. The objectives of the present study were to (1) measure serum AMH concentrations in female rabbits and investigate the value of AMH as a diagnostic tool to differentiate between spayed and intact does and (2) relate measured AMH levels to pseudopregnancy and ovarian follicle numbers. For AMH measurement, serum samples were obtained from sexually intact (n = 64) and spayed (n = 22) female rabbits. Spayed does were of various breeds; intact rabbits were Zika hybrid rabbits. In the intact does, AMH measurement was complemented by determination of progesterone levels, gynaecological examination and histopathological evaluation of the uterus and ovaries, including follicle counts. Serum AMH and progesterone concentrations were measured using a human-based chemiluminescence immunoassay (CLIA) and an enzyme-linked fluorescence assay (ELFA), respectively. Depending on progesterone levels, sexually intact does were classified into follicular (n = 52) or luteal phase (n = 12). Median serum AMH levels were 1.53 ng/ml (range 0.77-3.36 ng/ml) in intact and 0.06 ng/ml (range ≤0.01-0.23 ng/ml) in spayed does. AMH concentrations between the intact and spayed rabbits differed significantly and did not overlap (p < .001). Receiver operating characteristic (ROC) curve analysis yielded a sensitivity and specificity of 100% for a cut-off level of 0.50 ng/ml. Follicular or luteal phase had no significant influence on measured AMH levels (t = 0.061, df = 62, p = .951). While the number of secondary follicles correlated significantly with AMH concentrations (rs  = 0.410, p = .001), the number of primary or antral follicles did not (rs  = 0.241, p = .055 and rs  = 0.137, p = .281, respectively). In conclusion, a single determination of serum AMH concentrations was adequate to distinguish spayed from intact female rabbits. Among sexually intact individuals, whether does were in follicular or luteal phase had no significant influence on measured serum AMH concentrations. The relationship between small growing follicles and AMH levels as described in other species could be partially confirmed, as secondary follicles correlated significantly with AMH.

Acbp is essential for decidualization during early pregnancy in mice.

Decidualization of uterine stromal cells plays an important role in the establishment of normal pregnancy. Previous studies have demonstrated that Acyl-CoA binding protein (Acbp) is critical to cellular proliferation, differentiation, mitochondrial functions, and autophagy. The characterization and physiological function of Acbp during decidualization remain largely unknown. In the present study, we conducted the expression profile of Acbp in the endometrium of early pregnant mice. With the occurrence of decidualization, the expression of Acbp gradually increased. Similarly, Acbp expression was also strongly expressed in decidualized cells following artificial decidualization, both in vivo and in vitro. We applied the mice pseudopregnancy model to reveal that the expression of Acbp in the endometrium of early pregnant mice was not induced by embryonic signaling. Moreover, P4 significantly upregulated the expression of Acbp, whereas E2 appeared to have no regulating effect on Acbp expression in uterine stromal cells. Concurrently, we found that interfering with Acbp attenuated decidualization, and that might due to mitochondrial dysfunctions and the inhibition of fatty acid oxidation. The level of autophagy was increased after knocking down Acbp. During induced decidualization, the expression of ACBP was decreased with the treatment of rapamycin (an autophagy inducer), while increased with the addition of Chloroquine (an autophagy inhibitor). Our work suggests that Acbp plays an essential role in the proliferation and differentiation of stromal cells during decidualization through regulating mitochondrial functions, fatty acid oxidation, and autophagy.

Successful pseudopregnancy of rats by short period artificial stimulation using sonic vibration.

Psuedopregnancy for embryo transfer (ET) is usually induced in rats by mating with vasectomized males. Previously, we successfully induced pseudopregnancy using sonic vibration instead (Easy-ET method). The transferred embryos developed normally. Conventionally, stimulation is performed 7 × 30 s with 5 min intervals at the day before ET. However, this protocol is time-consuming because it imitates natural mating behavior. Here, we investigated pseudopregnancy induction with shorter stimulation times. Stimulation was performed 2 × 30 s, with 30 s intervals at the proestrus stage at the day before ET. Of the transferred pronuclear or two-cell embryos, 43% or 62% developed normally, respectively. Furthermore, 67% or 68% of transferred pronuclear or two-cell embryos in rats at estrus stage stimulated on the day of ET developed normally, respectively. Pseudopregnancy was successfully induced with shorter stimulation. Furthermore, this protocol may be used to perform a single-day stimulation and ET operation at the estrus stage.

HIF-1α Activation Promotes Luteolysis by Enhancing ROS Levels in the Corpus Luteum of Pseudopregnant Rats.

The increase of oxidative stress is one of the important characteristics of mammalian luteal regression. Previous investigations have revealed the essential role of reactive oxygen species (ROS) in luteal cell death during luteolysis, while it is unknown how ROS is regulated in this process. Considering the decrease of blood flow and increase of PGF2α during luteolysis, we hypothesized that the HIF-1α pathway may be involved in the regulation of ROS in the luteal cell of the late corpus luteum (CL). Here, by using a pseudopregnant rat model, we showed that the level of both HIF-1α and its downstream BNIP3 was increased during luteal regression. Consistently, we observed the increase of autophagy level during luteolysis, which is regulated in a Beclin1-independent manner. Comparing with early (Day 7 of pseudopregnancy) and middle CL (Day 14), the level of ROS was significantly increased in late CL, indicating the contribution of oxidative stress in luteolysis. Inhibition of HIF-1α by echinomycin (Ech), a potent HIF-1α inhibitor, ameliorated the upregulation of BNIP3 and NIX, as well as the induction of autophagy and the accumulation of ROS in luteal cells on Day 21 of pseudopregnancy. Morphologically, Ech treatment delayed the atrophy of the luteal structure at the late-luteal stage. An in vitro study indicated that inhibition of HIF-1α can also attenuate PGF2α -induced ROS and luteal cell apoptosis. Furthermore, the decrease of cell apoptosis can also be observed by ROS inhibition under PGF2α treatment. Taken together, our results indicated that HIF-1α signaling is involved in the regression of CL by modulating ROS production via orchestrating autophagy. Inhibition of HIF-1α could obviously hamper the apoptosis of luteal cells and the process of luteal regression.

Revisiting canine pseudocyesis.

Canine (Canis familiaris) pseudocyesis, pseudopregnancy, false pregnancy or nervous lactation is a frequent syndrome observed in non-pregnant, late diestrous or early anestrous females that is characterized by different degrees of mammary gland enlargement, maternal behavior and lactation. Further education about this frequent canine physiological event is still necessary to ensure optimal diagnosis and treatment strategies. Thus, the aim of this article was to review and update the physiopathology, physical and behavioral signs, diagnosis, treatment and prevention of pseudocyesis in bitches in which it is a clinical problem.

Induction of short-term pseudopregnancy in gilts by the administration of estradiol benzoate or estradiol dipropionate to achieve ovulatory synchronization for embryo collection.

A study was conducted to investigate whether ovulation in gilts could be synchronized for embryo collection by the administration of estradiol benzoate (EB) or estradiol dipropionate (EDP) to induce pseudopregnancy, followed by the treatment with prostaglandin F2α (PGF2α ) on 10 days after. Ten gilts each received a total of 20 mg of EB or EDP on Day 10 or EB on Day 10 and 14 to induce pseudopregnancy (Day 0 = onset of estrus). Donors received PGF2α 10 or 15 days (as a control) after the first administration of estrogens and subsequently eCG and hCG, and were then inseminated artificially. The embryos were collected 7 days after the administration of hCG, and assessed for embryo yield and their developmental stages. All protocols resulted in good embryo yield (9.8-13.2 embryos in average), and the embryos showed average ability to develop to the expanded blastocyst stage (3.29-4.03 as developmental scores) without any significant differences among the protocols. These results suggest that the administration of PGF2α 10 days after the treatment of gilts with EB or EDP would allow synchronization of ovulation and embryo collection, as well as shortening the period from estrus detection to embryo collection, thus improving embryo collection efficiency.

Replacement of surgical vasectomy through the use of wild-type sterile hybrids.

For the production and rederivation of mouse strains, pseudopregnant female mice are used for embryo transfer and serve as surrogate mothers to support embryo development to term. Vasectomized males are commonly used to render pseudopregnancy in females, generated by surgical procedures associated with considerable pain and discomfort. Genetically modified mouse strains with a sterility phenotype provide a non-surgical replacement and represent an important application of the 3Rs (Replacement, Reduction, Refinement). However, the maintenance of such genetically modified mouse strains requires extensive breeding and genotyping procedures, which are regulated procedures under national legislation. As an alternative, we have explored the use of sterile male hybrids that result when two wild-type mouse subspecies, Mus musculus musculus and Mus musculus domesticus, interbreed. We find the male STUSB6F1 hybrid, resulting from the mating of female STUS/Fore with male C57BL/6J, ideally suited and demonstrate that its performance for the production of oviduct and uterine transfer recipients is indistinguishable when compared to surgically vasectomized mice. The use of these sterile hybrids avoids the necessity for surgical procedures or the breeding of sterile genetically modified lines and can be generated by the simple mating of two wild-type laboratory strains-a non-regulated procedure. Furthermore, in contrast with the breeding of genetically sterile mice, all male offspring are sterile and suitable for the generation of pseudopregnancy, allowing their efficient production with minimal breeding pairs.

The Pre-Implantation Embryo Induces Uterine Inflammatory Reaction in Mice.

It has been well established that uterine function during the peri-implantation period is precisely regulated by ovarian estrogen and progesterone. The embryo enters the uterine cavity before implantation. However, the impact of pre-implantation embryo on uterine function is largely unknown. In the present study, we performed RNA-seq analysis of mouse uterus on day 4 morning of natural pregnancy (with embryos in the uterus) and pseudo-pregnancy (without embryos in the uterus). We found that 146 genes were upregulated, and 77 genes were downregulated by the pre-implantation embryo. Gene ontology and gene network analysis highlighted the activation of inflammatory reaction in the uterus. By examining the promoter region of differentially expressed genes, we found that NF-kappaB was a causal transcription factor. Finally, we validated 4 inflammation-related genes by quantitative RT-PCR. These 4 genes are likely the main mediators of the inflammatory reaction in the uterus triggered by the pre-implantation embryo. Our data indicated that the pre-implantation embryo causes uterine inflammatory reaction, which in turn might contribute to the establishment of uterine receptivity and embryo implantation.

Endocrine profiling of reproductive status and evidence of pseudopregnancy in the Pacific walrus (Odobenus rosmarus divergens).

Endocrine profiling is an increasingly utilized tool for detecting pregnancies in wild populations of mammals. Given the difficulty in calculating reproductive rates of Pacific walruses (Odobenus rosmarus divergens) the use of endocrine techniques for determining pregnancy rates could be particularly useful for management of the population. The goals of this study were to 1) determine if progesterone and total estrogen concentrations in ovarian tissues of female walruses could be used to determine reproductive state and 2) determine if walruses undergo a functional postpartum estrus, as is seen in other pinnipeds. Ovaries were collected from female walruses (n = 13) hunted in subsistence hunts by Alaska Native communities. Females were categorized as postpartum, full-term pregnant, pregnant diapause or unbred. Total estrogen concentrations were greatest in unbred (n = 2) and pregnant (n = 2) females. Progesterone concentrations were also nominally larger in unbred (n = 2) than pregnant (n = 2) and postpartum (n = 9) animals. Small samples sizes precluded the use of statistical comparisons among groups. Corpora lutea tissue samples in this study did not reflect the presence of a postpartum estrus in the month of May as postpartum females yielded lower total estrogen concentrations than unbred or pregnant animals. Both unbred animals were in a state of pseudopregnancy, which has not been physiologically described for this species before. The progesterone profiles in late (59 ng/g) and early (140 ng/g) pregnancy were lower than expected and fell within the range of the postpartum females (36-210 ng/g), suggesting low production of the hormone by the corpus luteum during these phases of pregnancy. Profiling reproductive hormones in free-ranging walruses demonstrates that an endocrine approach may be a valuable tool for determining reproductive status of females, however increased sample sizes and time of year must be considered to accurately separate pregnant versus pseudopregnant individuals.

Artificial lactation by exogenous hormone treatment in non-pregnant sows.

This study aimed to determine if lactation can be induced by exogenous hormonal treatment in non-pregnant sows. In experiment 1, pseudopregnant animals were divided into four groups and given: 1) 5 mg of estradiol dipropionate (EDP) 5 days before (n = 4), 2) 5 mg of EDP 10 days before (n = 3), 3) 10 mg of EDP 5 days before (n = 3) or 4) 10 mg of EDP 10 days (n = 3) before PGF2α treatment. Artificial lactation was induced in seven pseudopregnant sows (53.8%) by exogenous hormonal treatment. There was no significant effect of either an increased EDP dosage or interval from the EDP treatment to PGF2α treatment on the induction rate of artificial lactation. In experiment 2, milk samples were collected from artificial lactating and natural lactating sows (n = 6). IgG and IgA levels in the milk collected from both groups were significantly associated with time during the experimental period. Milk IgG levels 24 h after PGF2α treatment in artificial lactating sows were higher than those in the colostrum of lactating sows. In experiment 3, hormonal profiles in pseudopregnant sows with (n = 3) or without (n = 3) EDP treatment were determined. There was a significant difference in estradiol-17ß levels on days 8, 7 and 5 before PGF2α treatment between groups. Progesterone and prolactin concentrations did not differ between groups. The present study revealed for the first time that lactation could be induced by exogenous hormonal treatment in non-pregnant sows and that the milk collected from these sows contained high immunoglobulin levels.

Energy homeostasis in rabbit does during pregnancy and pseudopregnancy.

This study was conducted to evaluate the changing concentrations of metabolic hormones and metabolites in pregnant (P) and pseudopregnant (PP) rabbit does. Twenty-five New Zealand White rabbit does were submitted to artificial insemination (AI) and then classified as P (n = 15) or PP (n = 10). Blood samples were collected weekly until day 32 post AI. During pregnancy, leptin concentrations were greater on Days 14 and 21 (P < 0.05), while insulin was greater on days 21 and 32 post AI (P < 0.05) compared to PP does. The triiodothyronine/thyroxine (T3/T4) ratio was greater in the first and last week (P < 0.001); whereas, cortisol concentrations were greater in the last week of pregnancy and after parturition (P < 0.01) compared with that of PP does. Non-esterified fatty acids (NEFA) concentrations increased from day 7 until day 32 post AI (P < 0.05). Glucose concentrations were unchanged throughout pregnancy although concentrations were positively associated with litter size. These results indicate concentrations of hormones and metabolites change during pregnancy to ensure energy requirements are met for both the foetuses and the maternal tissues. Physiological hyperleptinemia, hyperinsulinemia, and changes in cortisol as well as thyroid hormones indicate there is an adaptation of metabolic functions induced by pregnancy. These adaptations could be mediated by gonadal steroids because changes mainly occur in the second half of pregnancy when the profile of the sex hormones differs between P and PP does.

Simple induction of pseudopregnancy by artificial stimulation using a sonic vibration in rats.

Embryo transfer has been used as one of the essential reproductive technologies for production of new strains and maintenance of genetic resources in animals. Mating with vasectomised male rats is a requirement for inducing pseudopregnancy in female rats selected for embryo transfer. Although this procedure has been used routinely, large breeding space and high expenditure are required to maintain a sufficient number of females and vasectomised males. This study was performed to induce pseudopregnancy in females by artificial stimulation using sonic vibration instead of vasectomised males. The females continued to be in the dioestrus stage for at least 14 days after artificial stimulation was performed. Of fresh 2-cell embryos that transferred into the oviducts of females after artificial stimulation, 56% was implanted and 50% was developed to offspring. Approximately 46% of the frozen 2-cell embryos were implanted and 24% developed into offspring. Furthermore, 66% of the fresh pronuclear embryos were implanted and 60% developed into offspring. This study successfully induced pseudopregnancy in rat females by artificial stimulation using a sonic vibration. This method, 'Easy-ET', was useful for efficient production and maintenance of rat strains.

ERα-dependent stimulation of LCN2 in uterine epithelium during mouse early pregnancy.

Maintenance of a suitable uterine milieu is important for embryo development and subsequent implantation during early pregnancy. High estrogen level in proestrous and estrous stages is essential for uterine anti-bacterial activity during preimplantation period. Lipocalin-2 is an essential molecule which prevents bacterial infection by sequestering iron. In this study, the highest expression of lipocalin-2 is observed in the endometrial epithelium on day 1 of normal pregnancy and pseudopregnancy, which exhibit a similar hormone scenario. By injecting the agonists for estrogen receptor α and estrogen receptor ß in ovariectomized mice, we found estrogen receptor α is the dominant member for estrogen regulation on lipocalin-2 expression. Estrogen treatment in estrogen receptor α-knockout mice further confirmed the role of estrogen receptor α. Using published data from whole-genome estrogen receptor α binding site assay, significant estrogen receptor α recruitment peaks are found at the downstream of lipocalin-2 gene after estrogen treatment. Furthermore, to study the anti-bacterial activity of lipocalin-2 in uterus, Escherichia coli is injected to mimic bacterial infection. Our results showed an obvious induction of lipocalin-2 in Escherichia coli-treated group. Taken together, this study indicates estrogen regulation of lipocalin-2 in uterine epithelium is mediated by estrogen receptor α, and lipocalin-2 may have anti-bacterial activity during early pregnancy.

Progesterone-induced RNA Hand2os1 regulates decidualization in mice uteri.

Decidualization is a critical process for successful embryo implantation and subsequent placenta formation. The characterization and physiological function of lncRNA during decidualization remain largely unknown. In the present study, we conducted RNA-sequencing analysis to compare gene expression between decidua of days 6 and 8, and normal pregnant endometrium (day 4). A total of 2332 high-confidence putative lncRNA transcripts were expressed. Functional clustering analysis of cis and trans lncRNA targets showed that differentially expressed lncRNAs may regulate multiple gene ontology terms and pathways that have important functions in decidualization. Subsequent analyses using qRT-PCR validated that eight of all lncRNAs were differentially regulated in mice uteri during decidualization, both in vivo and in vitro. Furthermore, we showed that differentially expressed lncRNA of Hand2os1 was specifically detected in stromal cells on days 2 to 5 of pregnancy and was strongly upregulated in decidual cells on days 6-8 of pregnancy. Similarly, Hand2os1 expression was also strongly expressed in decidualized cells following artificial decidualization, both in vivo and in vitro. In uterine stromal cells, P4 was able to significantly upregulate the expression of Hand2os1, but upregulation was impeded by RU486, whereas E2 appeared to have no regulating effect on Hand2os1 expression. Concurrently, Hand2os1 significantly promoted the decidual process in vitro and dramatically increased decidualization markers Prl8a2 and Prl3c1. Our results provide a valuable catalog for better understanding of the functional roles of lncRNAs in pregnant mouse uteri, as it relates to decidualization.