Potential roles for the kisspeptin/kisspeptin receptor system in implantation and placentation.

BACKGROUND: Initially identified as suppressors of metastasis in various types of cancer, kisspeptins are a family of neuropeptides that are key regulators of the mammalian reproductive axis. Accumulating evidence has shown that kisspeptin is able to control both the pulsatile and surge GnRH release, playing fundamental roles in female reproduction, which include the secretion of gonadotropins, puberty onset, brain sex differentiation, ovulation and the metabolic regulation of fertility. Furthermore, recent studies have demonstrated the involvement of the kisspeptin system in the processes of implantation and placentation. This review summarizes the current knowledge of the pathophysiological role and utility of these local placental regulatory factors as potential biomarkers during the early human gestation. OBJECTIVE AND RATIONALE: A successful pregnancy, from the initiation of embryo implantation to parturition, is a complex process that requires the orchestration of a series of events. This review aims to concisely summarize what is known about the role of the kisspeptin system in implantation, placentation, early human pregnancy and pregnancy-related disorders, and to develop strategies for predicting, diagnosing and treating these abnormalities. SEARCH METHODS: Using the PubMed and Google Scholar databases, we performed comprehensive literature searches in the English language describing the advancement of kisspeptins and the kisspeptin receptor (KISS1R) in implantation, placentation and early pregnancy in humans, since its initial identification in 1996 and ending in July 2018. OUTCOMES: Recent studies have shown the coordinated spatial and temporal expression patterns of kisspeptins and KISS1R during human pregnancy. The experimental data gathered recently suggest putative roles of kisspeptin signaling in the regulation of trophoblast invasion, embryo implantation, placentation and early pregnancy. Dysregulation of the kisspeptin system may negatively affect the processes of implantation as well as placentation. Clinical studies indicate that the circulating levels of kisspeptins or the expression levels of kisspeptin/KISS1R in the placental tissues may be used as potential diagnostic markers for women with miscarriage and gestational trophoblastic neoplasia. WIDER IMPLICATIONS: Comprehensive research on the pathophysiological role of the kisspeptin/KISS1R system in implantation and placentation will provide a dynamic and powerful approach to understanding the processes of early pregnancy, with potential applications in observational and analytic screening as well as the diagnosis, prognosis and treatment of implantation failure and early pregnancy-related disorders.

Assessment of growth and metabolism characteristics in offspring of dehydroepiandrosterone-induced polycystic ovary syndrome adults.

Polycystic ovary syndrome (PCOS) is a common reproductive disorder that has many characteristic features including hyperandrogenemia, insulin resistance and obesity, which may have significant implications for pregnancy outcomes and long-term health of women. Daughters born to PCOS mothers constitute a high-risk group for metabolic and reproductive derangements, but no report has described potential growth and metabolic risk factors for such female offspring. Hence, we used a mouse model of dehydroepiandrosterone (DHEA)-induced PCOS to study the mechanisms underlying the pathology of PCOS by investigating the growth, developmental characteristics, metabolic indexes and expression profiles of key genes of offspring born to the models. We found that the average litter size was significantly smaller in the DHEA group, and female offspring had sustained higher body weight, increased body fat and triglyceride content in serum and liver; they also exhibited decreased energy expenditure, oxygen consumption and impaired glucose tolerance. Genes related to glucolipid metabolism such as Pparγ, Acot1/2, Fgf21, Pdk4 and Inhbb were upregulated in the liver of the offspring in DHEA group compared with those in controls, whereas Cyp17a1 expression was significantly decreased. However, the expression of these genes was not detected in male offspring. Our results show that female offspring in DHEA group exhibit perturbed growth and glucolipid metabolism that were not observed in male offspring.

Role of Sirt3 in mitochondrial biogenesis and developmental competence of human in vitro matured oocytes.

STUDY QUESTION: Does Sirt3 dysfunction result in poor developmental outcomes for human oocytes after in vitro maturation (IVM)? SUMMARY ANSWER: Inefficient Sirt3 expression induced decreased mitochondrial DNA copy number and biogenesis, and therefore impaired the developmental competence of human IVM oocytes. WHAT IS KNOWN ALREADY: Cytoplasmic immaturity in IVM oocytes may lead to reduced developmental competence. Mitochondrial dysfunction results in the accumulation of free radicals and leads to DNA mutations, protein damage, telomere shortening and apoptosis. SIRT3 (in the Sirtuin protein family) has emerged as a mitochondrial fidelity protein that directs energy generation and regulates reactive oxygen species scavenging proteins. STUDY DESIGN, SIZE, DURATION: In vivo matured metaphase II (IVO-MII) oocytes and IVM-MII oocytes were donated by 324 infertile patients undergoing assisted reproductive technology cycles (12 patients for 60 IVO oocytes, and 312 patients for 403 IVM oocytes). Five oocytes each in the germinal vesicle (GV), IVM and IVO groups were compared with respect to mRNA levels for Sirt1-7 mRNA, and five samples at each developmental stage were analysed for Sirt3 mRNA. IVM-MII oocytes were injected with in vitro transcribed mRNA (n = 59) or small interfering RNA (siRNA) (n = 78). In human and mouse, IVM, mRNA-injection IVM, and siRNA-injection IVM groups (n = 5 each) were analysed for mitochondrial DNA copy number and abundance of Sirt3 and Pgc1α (an inducer of mitochondrial biogenesis) mRNAs. Human blastocysts in the IVO (n = 12), IVM (n = 9), mRNA-injection IVM (n = 13) and siRNA-injection IVM (n = 6) groups were used to generate embryonic stem cells (ESCs). In addition, 587 IVO-MII and 1737 IVM-MII oocytes from 83 mice were collected to compare the preliminary human oocyte data with another species. PARTICIPANTS/MATERIALS, SETTING, METHODS: mRNA abundance was analysed by single-cell real-time PCR. Karyotyping of human embryos was performed with an array comparative genomic hybridization method, and that of ESCs by cytogenetic analysis. The function of the Sirt3 gene was investigated using siRNA and in vitro transcribed mRNA injection. Markers of ESCs were identified using immunofluorescence. MAIN RESULTS AND THE ROLE OF CHANCE: A retrospective analysis revealed a higher spontaneous abortion rate (P < 0.01) and decrease in high quality embryo rate (P < 0.01) in patients with IVM versus controlled ovarian stimulation (COS) cycles. A decrease in abundance of Sirt3 mRNA (P < 0.01) and mitochondrial biogenesis (P < 0.05) were identified in human IVM compared with IVO oocytes. The developmental potential of human IVM-MII oocytes to the blastocyst stage was significantly reduced when Sirt3 mRNA was inhibited by siRNA (P < 0.05 versus IVM-MII group) but could be up-regulated by injection of Sirt3 mRNAs. Compared with IVO-MII group, comparable generation efficiency of human ESCs can be obtained using blastocysts from IVM-MII oocytes with Sirt3 mRNA injection. Sirt3 mRNA was significantly increased in mouse zygotes after IVF (P < 0.001 versus MII oocytes) but gradually declined until the blastocyst stage. In mice, lower Sirt3 mRNA levels were observed IVM-MII oocytes and preimplantation embryos compared with in vivo controls, and mitochondrial biogenesis and the developmental efficiency from oocytes to blastocyst were affected by the abundance of Sirt3 mRNA in accordance with human. Therefore a similar role for Sirt3 mRNA in IVM-MII oocytes was observed in mouse and human. LIMITATIONS, REASONS FOR CAUTION: The couples in the study had a variety of different simple and complex factors causing infertility. Additional studies with a larger number of oocytes are required to confirm the present results owing to the limited number of human oocytes in the present study. WIDER IMPLICATIONS OF THE FINDINGS: To our knowledge, this is the first study investigating a role of the Sirt3 gene in mitochondrial biogenesis and the developmental competence of human IVM-MII oocytes. The observation may help to improve clinical application of the IVM procedure. STUDY FUNDING/COMPETING INTERESTS: This work was supported in part by the National Natural Science Foundation of Key Program (31230047), Ministry of Science and Technology of China Grants (973 program; 2014CB943203), the National Natural Science Foundation of General Program (31371521 and 81571400), Beijing Nova Program (xxjh2015011), and Specialized Research Fund for the Doctoral Program of Higher Education (20120001130008) and the National Natural Science Foundation of Young Scholar (31501201). The authors have declared that no conflict of interest exists.

Ascorbic acid improves pluripotency of human parthenogenetic embryonic stem cells through modifying imprinted gene expression in the Dlk1-Dio3 region.

INTRODUCTION: Human parthenogenetic embryonic stem cells (hpESCs) are generated from artificially activated oocytes, however, the issue of whether hpESCs have equivalent differentiation ability to human fertilized embryonic stem cells remains controversial. METHODS: hpESCs were injected into male severe combined immunodeficiency (SCID) mice and the efficiency of teratoma formation was calculated. Then the gene expression and methylation modification were detected by real time-PCR and bisulfate methods. RESULTS: Comparison of five hpESCs with different differentiation abilities revealed that levels of paternal genes in the Dlk1-Dio3 region on chromosome 14 in the hpESCs with high differentiation potential are enhanced, but strictly methylated and silenced in the hpESCs with lower differentiation potential. Treatment with ascorbic acid, rescued their ability to support teratoma formation and altered the expression profiles of paternally expressed genes in hpESCs that could not form teratoma easily. No differences in the expression of other imprinting genes were evident between hpESCs with higher and lower differentiation potential, except for those in the Dlk1-Dio3 region. CONCLUSIONS: The Dlk1-Dio3 imprinting gene cluster distinguishes the differentiation ability of hpESCs. Moreover, modification by ascorbic acid may facilitate application of hpESCs to clinical settings in the future by enhancing their pluripotency.

Aberrant expression of maternal Plk1 and Dctn3 results in the developmental failure of human in-vivo- and in-vitro-matured oocytes.

Fertilisation is the first step in embryonic development, and dynamic changes of key genes may potentially improve assisted reproduction techniques efficiency during this process. Here, we analysed genes that were differentially expressed between oocytes and zygotes and focused on cytokinesis-related genes. Plk1 and Dctn3 were identified as showing dramatic changes in expression during fertilisation and were suggested to play a key role in inducing aneuploidy in zygotes and 8-cell embryos. Moreover, we found that maternal Plk1 and Dctn3 were expressed at lower levels in in vitro matured oocytes, which may have contributed to the high ratio of resulting embryos with abnormal Plk1 and Dctn3 expression levels, thereby reducing the developmental competence of the resulting embryos. Furthermore, the overexpression of Dctn3 can silence Plk1 expression, which suggests a potential regulation mechanism. In conclusion, our present study showed that aberrant expression of Plk1 and Dctn3 increases embryo aneuploidy and developmental failure, particularly in in vitro matured oocytes. Our results facilitate a better understanding of the effects of oocyte maternal gene expression on embryonic development and can be used to improve the outcome of assisted reproduction techniques.

Chromosomal aberrations in in-vitro matured oocytes influence implantation and ongoing pregnancy rates in a mouse model undergoing intracytoplasmic sperm injection.

Implantation failure and early pregnancy loss have been reported to be closely related to the quality of mammalian oocytes; however, the pregnant outcome of embryos from in-vitro matured (IVM) oocytes remains unknown. In this study we examined spindle assembly and chromosome segregation during differentiation, and the duration of IVM of mouse oocytes. The resulting implantation and pregnancy outcomes were analyzed to clarify the relationship between the spindle and chromosomes of IVM oocytes and implantation and early pregnancy. Cumulus-enclosed germinal vesicle oocytes were collected and randomly cultured in IVM medium with different IVM durations. One part of IVM oocytes were analyzed the spindle and chromosome morphology by immunofluorescence method, and the other part of them were fertilized by intracytoplasmic sperm injection. The resulting embryos were transferred into pseudo-pregnant female mice, and the post-implantation and full term development was observed. The chromosome aberrations and incorrect spindle assembly seems not affect the early development and blastocyst cell number derived from IVM oocytes, however the development potential of the resulting embryos after implantation were significant decreased with the ratio increasing of chromosome aberrations and incorrect spindle assembly. Accordingly, the full-term development was also decreased. In conclusion, the present study showed the spindle assembly of in vitro-matured oocytes was one of the most important factors that affected the implantation and ongoing pregnancy rates of IVM oocytes, and the improvement by an appropriate duration of maturation in vitro will enhance the post-implantation development potential of the resulting embryos, and decrease implantation failure and early pregnancy loss.

Gonadotropin-mediated dynamic alterations during bovine oocyte maturation in vitro.

Gonadotropins have been widely used in human-assisted reproduction and animal science for the past four decades. However, the effects of gonadotropins on oocyte maturation at the molecular and biochemical levels are poorly understood. To determine the effects of gonadotropins (recombinant follicle stimulating hormone and urinary human menopausal gonadotropin) on oocyte maturation, we used the bovine oocyte in vitro maturation model. First, we studied the effects of increasing gonadotropin concentrations on nuclear maturation and mitochondrial function in oocytes. Gonadotropins at concentrations of 0.075 and 0.75 IU/ml improved nuclear maturation and increased inner mitochondrial membrane potential and ATP levels; however, there were no beneficial effects at concentrations of 7.5 and 75 IU/ml. Second, we studied the effects of increasing gonadotropin concentrations on the status of methylation in matured (MII) oocytes. Aberrant methylation and demethylation of H19, SNRPN, and PEG3 genes were observed in MII oocytes at all concentrations except 0.075 IU/ml. The expression of genes that function in spindle formation, cell cycle control, and methylation was also downregulated by high gonadotropin concentrations. In conclusion, we established the optimal gonadotropin concentration (i.e., 0.075 IU/ml) to be used for bovine oocyte in vitro maturation studies. These results may provide a guide for clinical stimulation protocols and help to reduce the risks associated with gonadotropin administration during in vitro fertilization treatment.

Improved efficiency of microsurgical enucleated tripronuclear zygotes development and embryonic stem cell derivation by supplementing epidermal growth factor, brain-derived neurotrophic factor, and insulin-like growth factor-1.

Human embryonic stem cells (hESCs) hold great promise for future clinical cell therapies because of their unique potential to differentiate into all human cell types. However, the destruction of normal fertilized embryos and the derivation of hESCs for research has resulted in polarized ethical debates, with most of the controversy centered on embryo destruction. Therefore, due to less ethical controversy surrounding them, abnormal fertilized zygotes that are usually discarded are a potential feasible resource for the derivation of hESCs. Microsurgery on human polyspermic zygotes can contribute to the derivation of hESCs, but the efficiency is much lower. Here, we reported a culture system to enhance the developmental competence of such microsurgical human polyspermic zygotes by EGF-BDNF-IGF-1 combination, which eventually resulted in the increased derivation efficiency of hESCs from them. We found that the developmental efficiency of microsurgical enucleated tripronuclear (3PN) embryos cultured with the EGF-BDNF-IGF-1 combination was significantly increased compared with the control group. More importantly, when the microsurgical enucleated 3PN embryos were cultured in medium supplemented with EGF-BDNF-IGF-1, the frequency ratio of chromosome abnormality was reduced. Our present study will facilitate the development of hESC line derivation in subsequent studies and also provide an additional choice for infertile couples.

Aberrant epigenetic modification in murine brain tissues of offspring from preimplantation genetic diagnosis blastomere biopsies.

Preimplantation genetic diagnosis (PGD) has been prevalent in the field of assisted reproductive technology, yet the long-term risks of PGD to offspring remain unknown. In the present study, the early development of PGD embryos, postimplantation characteristics, and birth rate following PGD were determined. Moreover, the behavior of the offspring conceived from the biopsied embryos was evaluated with the Morris water maze and pole climbing tests. Finally, the epigenetic modification of the global genome and methylation patterns for the H19, Igf2, and Snrpn imprinted genes were identified. The results indicated a significant delay in the blastocoel formation of PGD embryos and a decrease in the implantation ability of these embryos, which was related to the decreased number of cells in the PGD blastocysts. The PGD mice spent more time on both the nontrained quadrant of the water maze and climbing down the pole. Furthermore, the 5-hydroxymethylcytosine content in the brain tissues of PGD mice was significantly increased, but no difference was found in 5-methylcytosine content. The differentially methylated regions of H19/Igf2 exhibited decreased methylation patterns, but that of Snrpn was normal, compared to the control group. Quantitative RT-PCR indicated that Igf2 mRNA expression was significantly decreased but that H19 and Snrpn mRNAs were expressed normally. In conclusion, blastomere biopsies in PGD procedures carry potential risks to embryo development and the behavior of resulting offspring; these risks may arise from aberrant epigenetic modification and methylation patterns in brain tissues. Further studies are needed to better understand the risks associated with PGD.

Improvement in the quality of heterotopic allotransplanted mouse ovarian tissues with basic fibroblast growth factor and fibrin hydrogel.

STUDY QUESTION: Does basic fibroblast growth factor (bFGF) in combination with fibrin hydrogel improve follicle development and revascularization of heterotopically transplanted mouse ovarian tissues? SUMMARY ANSWER: Treatment of transplanted ovarian tissues with higher concentrations (75, 100 and 150 µg/ml), but not lower concentrations (25 and 50 µg/ml), of bFGF significantly improved primordial follicle survival and angiogenesis, while apoptosis of follicles and stromal cells was significantly decreased. WHAT IS KNOWN ALREADY: Use of transplanted ovarian tissues in female fertility preservation is limited by the massive loss of follicles and ischemia-reperfusion injury due to the expected delay in revascularization. STUDY DESIGN, SIZE AND DURATION: Ovarian tissues from 18-day-old ICR mice were encapsulated in fibrin hydrogel mixed with different concentrations of bFGF, then transplanted under the skin of adult female mice for 1 week. The ovarian tissues treated without fibrin hydrogels and bFGF were designated as Control group I, and the ovarian tissues treated with fibrin hydrogels but without bFGF were designated as Control group II. The ovarian tissues treated with 25 and 50 µg/ml bFGF were designated as the lower concentration group, and the ovarian tissues treated with 75, 100 and 150 µg/ml bFGF were designated as the higher concentration group. MATERIALS, SETTING AND METHODS: Assessment of follicular quantity and follicle classification was carried out by histologic analysis. Follicle proliferation was evidenced by immunostaining with proliferating cell nuclear antigen and apoptosis was verified by anti-active caspase-3 staining. Epithelial cells of new blood vessels were stained using CD31 antibody to evaluate neoangiogenesis, and the blood vessel density was analyzed by immunohistochemistry. MAIN RESULTS AND THE ROLE OF CHANCE: The ovarian tissues were recovered 1 week post-transplantation. Compared with the control group, the survival and proliferation of the follicles was significantly increased, the apoptosis of follicles and stromal cells was significantly decreased, and angiogenesis was significantly enhanced when the transplanted ovarian tissues were treated with a higher concentration of bFGF. Treatment with a lower concentration of bFGF did not improve follicle survival and blood revascularization. LIMITATIONS, REASONS FOR CAUTION: The results obtained may not be fully extrapolated to humans because of the physiologic differences between mice and humans. WIDER IMPLICATIONS OF THE FINDINGS: For the first time, the present study investigated the role of bFGF in transplanted ovarian tissues and demonstrated that bFGF might significantly improve the quality of transplanted ovarian tissues by increasing follicle quantity and promoting neoangiogenesis. This study sets the stage for further study and application of ovarian tissue transplantation in clinics, and may eventually benefit females for fertility preservation. STUDY FUNDING/COMPETING INTEREST(S): This work was partially supported by the Ministry of Science and Technology of China Grants (973 Program; 2011CB944503 to Q.J.), the Program for Changjiang Scholars and Innovative Research Team in University of Ministry of Education of China (30825038 to Q.J.), and the National Natural Science Funds for Young Scholar (81200470 to Y.J. and 81000275 to Y.L.Y.). None of the authors have any conflicts of interest.