Exposure to BaA inhibits trophoblast cell invasion and induces miscarriage by regulating the DEC1/ARHGAP5 axis and promoting ubiquitination-mediated degradation of MMP2.

Benz[a]anthracene (BaA), a hazardous polycyclic aromatic hydrocarbon classified by the EPA, is a probable reproductive toxicant. Epidemiological studies suggest that BaA exposure may be a risk factor for recurrent miscarriage (RM). However, the underlying mechanisms are not well understood. This study identified DEC1 as a key gene through RNA-seq and single-cell RNA sequencing analysis. DEC1 expression was found to be downregulated in villous tissues from women with RM and in primary extravillous trophoblasts (EVTs) exposed to BaA. BaA suppressed DEC1 expression by promoting abnormal methylation patterns. Further analysis revealed that ARHGAP5 is a direct target of DEC1 in EVTs, where DEC1 inhibits trophoblast invasion by directly regulating ARHGAP5 transcription. Additionally, BaA destabilized matrix metalloproteinase 2 (MMP2) by activating the aryl hydrocarbon receptor (AhR) and promoting E3 ubiquitin ligase MID1-mediated degradation. In a mouse model, BaA induced miscarriage by modulating the DEC1/ARHGAP5 and MID1/MMP2 axes. Notably, BaA-induced miscarriage in mice was prevented by DEC1 overexpression or MID1 knockdown. These findings indicate that BaA exposure leads to miscarriage by suppressing the DEC1/ARHGAP5 pathway and enhancing the MID1/MMP2 pathway in human EVTs.

Cocktail treatment by GnRH-antagonist, letrozole, and mifepristone for the prevention of ovarian hyperstimulation syndrome: a prospective randomized trial.

OBJECTIVE: This study is aimed to determine the efficacy of a cocktail style treatment by combining GnRH-antagonist, letrozole, and mifepristone on the prevention of ovarian hyperstimulation syndrome (OHSS) in high-risk women. METHODS: This prospective, randomized controlled clinical trial was performed between January 2018 and December 2018. A total of 170 women who identified as high risk of OHSS during the ovarian hyperstimulation and underwent cryopreservation of whole embryos. On the day of oocyte retrieval, the combination group received 0.25 mg Cetrorelix for 3 d, 5 mg letrozole for 5 d, and 50 mg mifepristone for 3 d, the mifepristone group received 50 mg mifepristone for 3 d. A total of 156 cases were included in final analysis. All the frozen embryo transfer (FET) cycles were followed up until December 2021. RESULTS: The combination group showed significantly decreased incidence of moderate and severe OHSS than mifepristone group (20.5% vs. 42.3%), with remarkably reduced serum estradiol level on hCG + 3 and + 5 d, decreased ovarian diameter, and shortened luteal phase. Oocyte retrieval number, levels of estradiol on hCG + 0 and VEGF, and ovarian diameter on hCG + 5 were associated with the severity of the symptoms. There was no significant difference in cumulative live birth rates (LBRs) between the combination and mifepristone group (74.4% vs. 76.9%). CONCLUSIONS: The combination treatment effectively reduces the incidence of moderate/severe OHSS in high-risk women.

Cycle Characteristics and Pregnancy Outcomes of Early Rescue Intracytoplasmic Sperm Injection Cycles in Normal and Hyper-Ovarian Response Women: A Six-Year Retrospective Study.

This study aims to analyze the cycle characteristics, pregnancy, and neonatal outcomes in early rescue intracytoplasmic sperm injection (r-ICSI) cycles in normal and hyper-ovarian response women in their first IVF/ICSI attempts. Data from short-term in vitro fertilization (IVF, N = 7148), early r-ICSI (N = 618), and ICSI (N = 1744) cycles were retrospectively analyzed from normal and hyper-ovarian women who underwent their first IVF/ICSI cycles at our center from October 2015 to October 2021. The r-ICSI group was subdivided into partial r-ICSI (N = 451) and total r-ICSI (N = 167) based on the number of fertilized oocytes in the IVF part. Cyclic characteristics, pregnancy, delivery and neonatal outcomes in the fresh cycle were compared among the four groups; pregnancy, delivery and neonatal outcomes in frozen-thawed cycles were compared regarding cleavage and blastocyst transfers derived from r-ICSI cycles. Partial r-ICSI cycles showed different cyclic characteristics compared to total r-ICSI cycles, presenting as elevated AMH and estradiol levels on trigger day and an increased number of oocytes retrieved. Early r-ICSI delayed blastocyst development as seen by the increase in the number of day 6 blastocysts. There was no significant difference among the groups in clinical pregnancy, pregnancy loss, and live birth in fresh cleavage-stage embryo transfer cycles. However, early r-ICSI groups showed a reduction in clinical pregnancy and live birth rates in fresh blastocyst transfer cycles but not in the frozen-thawed cycles. For pregnant women, early r-ICSI did not show a negative effect on the risk of preterm birth, Cesarean section, neonatal birth weight, and sex ratio. In conclusion, early r-ICSI had comparable pregnancy, delivery, and neonatal outcomes when compared with short-term IVF and ICSI groups in fresh cleavage-stage embryo transfer cycles, but early r-ICSI did result in reduced pregnancy outcomes in fresh blastocyst embryo cycles, possibly due to delayed blastocyst development and asynchronization with the endometrium.

ADAD2 interacts with RNF17 in P-bodies to repress the Ping-pong cycle in pachytene piRNA biogenesis.

Pachytene piRNA biogenesis is a hallmark of the germline, distinct from another wave of pre-pachytene piRNA biogenesis with regard to the lack of a secondary amplification process known as the Ping-pong cycle. However, the underlying molecular mechanism and the venue for the suppression of the Ping-pong cycle remain elusive. Here, we showed that a testis-specific protein, ADAD2, interacts with a TDRD family member protein RNF17 and is associated with P-bodies. Importantly, ADAD2 directs RNF17 to repress Ping-pong activity in pachytene piRNA biogenesis. The P-body localization of RNF17 requires the intrinsically disordered domain of ADAD2. Deletion of Adad2 or Rnf17 causes the mislocalization of each other and subsequent Ping-pong activity derepression, secondary piRNAs overproduced, and disruption of P-body integrity at the meiotic stage, thereby leading to spermatogenesis arrested at the round spermatid stage. Collectively, by identifying the ADAD2-dependent mechanism, our study reveals a novel function of P-bodies in suppressing Ping-pong activity in pachytene piRNA biogenesis.

Comparison of clinical effects between early follicular prolonged GnRH agonist protocol and GnRH antagonist protocol in 3310 cycles: a retrospective study.

BACKGROUND: It is the duty of doctors to choose a safe, simple, economic and effective controlled ovulation stimulation (COS) protocol for the patients. This study aims to compare the clinical effects of the early follicular prolonged GnRH agonist (EFPL) and GnRH antagonist (GnRH-Ant) protocols, hoping to provide some reference for clinicians when choosing COS program. METHODS: A retrospective study included 3310 ovum pick up cycles undergoing assisted reproductive technology during January 2019 to May 2022 in Renmin Hospital of Wuhan University. Propensity Score Matching (PSM) and multivariable logistic regression analysis were used to improve the comparability between the two protocols. Subgroups were divided according to age, body mass index (BMI) and anti-Mullerian hormone (AMH). The live birth rate (LBR) and clinical pregnancy rate (CPR) were the primary outcomes. RESULTS: After PSM, the endometrial thickness, fresh embryo transplantation rate, chemical pregnancy rate, CPR were significantly higher in EFPL group than that in GnRH-Ant group (P < 0.001). The E2, LH, P values on trigger day were significantly lower in EFPL group (P < 0.001). The cycle cancellation rate was significantly reduced in EFPL group (P < 0.001). However, the total amount of Gn and duration of Gn were significantly increased in the EFPL group (P < 0.001). Multivariable logistic regression analysis showed that the LBR was significantly higher in EFPL group after matching [OR (95%CI), 1.86 (1.13, 3.05), P = 0.02], especially for those with age < 35 years [OR (95%CI), 1.95 (1.14, 3.34), P = 0.02], BMI < 24 kg/m2 [OR (95%CI), 2.08 (1.14, 3.80), P = 0.02], AMH levels ≥ 4.5 ng/ml [OR (95%CI), 4.19 (1.53, 11.43), P < 0.01]. CONCLUSION: EFPL regimen is more suitable to elicit live birth for those young patients with BMI < 24 kg/m2 and AMH ≥ 4.5 ng/ml. However, for patients with decreased ovarian reserve or advanced age, EFPL regimen has no advantage over the GnRH-Ant regimen.

Chromosomal analysis of 262 miscarried conceptuses: a retrospective study.

BACKGROUND: Embryonic chromosomal abnormality is one of the significant causative factors of pregnancy loss. Our goal was to investigate the differences of chromosomal abnormality between different conception modes in miscarried products of conception (POCs). METHODS: A retrospective study included 262 miscarried POCs from 167 women undergoing assisted reproductive treatment (ART) and 95 spontaneous pregnant (SP) women during March 2019 to March 2022 in Renmin Hospital of Wuhan University. Subgroups were divided according to age, fertilization method, types and stages of embryo transfer. The profiles of cytogenetic abnormalities in the miscarried POCs were measured via next-generation sequencing. RESULTS: The rate of chromosomal abnormality in the fresh embryo transfer group and the cleavage embryo transfer group was significantly higher than that in the frozen embryo transfer group (79.2% vs. 36%, P = 0.0001) and the blastocyst transfer group (66.7% vs. 32.1%, P = 0.0001) respectively. There was no significant difference in the rate of chromosomal abnormalities when compared by maternal age (49.2% vs. 62%, P = 0.066), types of conception (49.7% vs. 57.9%, P = 0.202), fertilization method (49.6% vs. 48.7%, P = 0.927) and frequency of abortion (56% vs. 47.6%, P = 0.183). However, the women aged ≥ 35 years had more frequent numerical abnormality (P = 0.002); patients using assisted reproductive technology had more rate of chromosomal structural abnormalities (26.5% vs. 7.3%, P = 0.005); the ICSI fertilization group has more frequency of deletion/microdeletion than the IVF fertilization group (80% vs. 31.3%, P = 0.019). CONCLUSION: Blastocyst transfer might help to reduce the incidence of miscarriage. In addition, "freezing all" should be considered if encountered hyper ovarian stimulation, to avoid the negative effect of high estrogen environment on embryo development. The higher incidence of structural abnormalities in miscarried POCs from assisted reproductive patients reminds us to pay attention to the safety of the technology for offspring.

Regulatory Effects of Fyn on Trophoblast Cell Behaviors and Function.

Fyn has been proven to be involved in various cell behaviors and pathophysiological processes. However, the expression and roles of Fyn in trophoblasts remain unclear. Here, we aimed to evaluate the participation of Fyn in trophoblast behavior and function, and the related mechanisms were briefly explored. Fyn expression in the HTR-8/SVneo, JEG-3, and JAR cell lines was evaluated by immunofluorescence, quantitative real-time PCR and western blotting. Fyn expression in human hydatidiform moles was also determined by immunohistochemistry and western blot. To explore the effects of Fyn, HTR-8/SVneo and JEG-3 cells were transfected with Fyn shRNA or overexpression plasmid or treated with the Fyn activity inhibitor SU6656 or ERK1/2 inhibitor U0126. The migration, proliferation, and apoptosis of trophoblast cells were assessed using transwell assays, flow cytometry, and cell counting kit-8 assays, respectively. The production of primary inflammatory cytokines, HLA-G and active matrix metallopeptidase (MMP) 2/9, and the phosphorylation of ERK1/2 and STAT3 were evaluated by ELISA, western blot, or gelatin zymography. The results showed that Fyn was expressed by trophoblast cells, mainly in the cytoplasm and membrane. Fyn expression and activity levels both increased in order from HTR-8/SVneo and JAR to JEG-3. The overexpression of Fyn promoted the proliferation and migration of trophoblast cells and inhibited their apoptosis, while the opposite effects were observed for Fyn knockdown and inhibition. Fyn regulated inflammatory cytokine production in trophoblast cells by promoting TGF-ß and IL-4 secretion while inhibiting IFN-γ and TNF-α secretion. Moreover, HLA-G expression in JEG-3 was positively regulated by Fyn. Fyn also facilitated the expression of active MMP2/9 and the activation of ERK1/2 and STAT3. Besides, it was confirmed that Fyn regulated trophoblast cell activities through ERK1/2 signal pathway by using U0126. Our study first detected the expression of Fyn in trophoblast cells. Fyn played pivotal roles in trophoblast cell behaviors and function, ERK1/2 was one of its targets, and MMP2/9 and STAT3 may also be involved in the regulatory mechanism.

Effect of GnRH-antagonist, mifepristone and letrozole on preventing ovarian hyperstimulation syndrome in rat model.

RESEARCH QUESTION: Can luteolysis-targeted drugs, gonadotrophin-releasing hormone antagonist (GnRH-ant), mifepristone and letrozole, administered separately or in combination, prevent the progression of ovarian hyperstimulation syndrome (OHSS) in a rat model? DESIGN: Thirty-six female Wistar rats were randomly divided into six groups, including control group (OHSS group, ovarian hyperstimulation-induced OHSS); GnRH-ant group (OHSS with GnRH-ant treatment); mifepristone group (OHSS with mifepristone treatment); letrozole group (OHSS with letrozole treatment); combination group (OHSS with GnRH-ant, mifepristone and letrozole treatment in combination). The main outcomes were the alterations in OHSS-related indices, including ovarian weight, vascular permeability, serum oestradiol and progesterone levels, corpus luteum proportion and diameter, ovarian vascular endothelial growth factor (VEGF), interleukin 6 (IL-6), caspase-3 and cleaved caspase-3 levels. RESULTS: No significant difference was found in body weight gain among the six groups. Compared with the control group, the OHSS group showed significant increases in all OHSS-related indices. GnRH-ant treatment showed decreases in vascular permeability, serum oestradiol level, corpus luteum diameter, ovarian VEGF /IL-6 mRNA levels, and increases in ovarian caspase-3 and cleaved caspase-3 levels. Mifepristone treatment demonstrated reduction in serum progesterone level and corpus luteum diameter, and elevation in ovarian caspase-3 and cleaved caspase-3 levels. Letrozole treatment displayed a decline in serum oestradiol level and corpus luteum diameter, and up-regulation in ovarian caspase-3 and cleaved caspase-3 levels. The combination treatment by GnRH-ant, mifepristone and letrozole showed enhanced synergistic effect on reducing OHSS-related indices. CONCLUSIONS: GnRH-ant, mifepristone and letrozole are beneficial in preventing the progression of OHSS through different luteolytic mechanisms. Cocktail style treatment shows enhanced synergistic effect on preventing the progression of OHSS.

Effects of artificial cycles with and without gonadotropin-releasing hormone agonist pretreatment on frozen embryo transfer outcomes.

OBJECTIVE: This study aimed to compare the pregnancy outcomes between women receiving frozen embryo transfer (FET) with hormone replacement treatment (HRT) with and without gonadotropin-releasing hormone agonist (GnRHa) pretreatment. METHODS: All consecutive women undergoing HRT cycles (2936 cycles) or HRT with GnRHa pretreatment (HRT + GnRHa, 303 cycles) at our reproductive center between January 2015 and December 2017 were analyzed retrospectively. RESULTS: The average age was higher in the HRT + GnRHa compared with the HRT group (34.0 ± 4.8 vs. 31.3 ± 4.4). However, the pregnancy outcomes were comparable between the two groups. The clinical pregnancy rate was significantly increased in younger women (≤35 years) in the HRT + GnRHa group compared with the HRT group (56.8% vs. 48.7%), but the live birth rates were similar in the two groups (44.2% vs. 38.4%). The HRT + GnRHa protocol significantly increased the clinical pregnancy rate (55.6% vs. 43.2%) and live birth rate (43.5% vs. 33.5%) compared with the HRT group among women with endometriosis, and significantly decreased the abortion rate in women with polycystic ovarian syndrome (3.1% vs. 16.4%). CONCLUSIONS: GnRHa pretreatment may improve pregnancy outcomes in women with endometriosis and polycystic ovarian syndrome.

The effects of methotrexate and uterine arterial embolization in patients with cesarean scar pregnancy: A retrospective case-control study.

In this study, we explored the optimal treatment for cesarean scar pregnancy (CSP). One hundred three women diagnosed with CSP received 1 of the 3 treatments: local or systemic methotrexate (MTX) injection and surgery (MTX + Surg), uterine arterial embolization (UAE) and surgery (UAE + Surg) or surgery only (Surg only). We compared their therapeutic effects and their follow-up results. There was no significant difference between the groups in the baseline of clinical characteristic except for the initial ß human chorionic gonadotropin levels, which was highest in the MTX + Surg group (median, [interquartile range]), (120,004 [16,720-181,727] mIU/mL), compared to the UAE + Surg group (38,219 [23,194-100,029] mIU/mL) and Surg only group (22,557 [9113-49,573] mIU/mL). There was no significant difference between groups in the sonographic characteristic of patients. The intraoperative hemorrhage was highest in the Surg-only group (7/42, 16.67%), compared to the MTX + Surg group (4/26, 15.38%) and the UAE + Surg group (0/35, 0%). The incidence of intrauterine adhesions was highest in the UAE + Surg group (20%), compared to the MTX + Surg group (0%) and the Surg only group (0%). The incidence of embryo residue was highest in Surg-only group (21.43%), compared to the MTX + Surg group (0%) and the UAE + Surg group (2.86%). To conclude, MTX injection plus surgery might be the best treatment for CSP patients.

Inhibition of TMEM16A impedes embryo implantation and decidualization in mice.

Recent studies revealed that TMEM16A is involved in several reproductive processes, including ovarian estrogen secretion and ovulation, sperm motility and acrosome reaction, fertilization and myometrium contraction. However, little is known about the expression and function of TMEM16A in embryo implantation and decidualization. In this study, we focused on the expression and regulation of TMEM16A in mouse uterus during early pregnancy. We found that TMEM16A is upregulated in uterine endometrium in response to embryo implantation and decidualization. Progesterone treatment could induce TMEM16A expression in endometrial stromal cells through progesterone receptor/c-Myc pathway, which is blocked by progesterone receptor antagonist or the inhibitor of c-Myc signaling pathway. Inhibition of TMEM16A by small molecule inhibitor (T16Ainh-A01) resulted in impaired embryo implantation and decidualization in mice. Treatment with either specific siRNA of Tmem16a or T16Ainh-A01 inhibited the decidualization and proliferation of mouse endometrial stromal cells. In conclusion, our results revealed that TMEM16A is involved in embryo implantation and decidualization in mice, compromised function of TMEM16A may lead to impaired embryo implantation and decidualization.

Effect of Progestin-primed Ovarian Stimulation Protocol on Outcomes of Aged Infertile Women Who Failed to Get Pregnant in the First IVF/ ICSI Cycle: A Self-controlled Study.

This study aimed to explore the outcomes of progestin-primed ovarian stimulation protocol (PPOS) in aged infertile women who failed to get pregnant in the first IVF/ICSI-ET cycles with GnRH-a long protocol. A self-controlled study was conducted to retrospectively investigate the clinical outcomes of 104 aged infertile patients who didn't get pregnant in the first IVF/ICSI-ET treatment by stimulating with GnRH-a long protocol (non-PPOS group), and underwent PPOS protocol (PPOS group) in the second cycle between January 2016 and December 2016 in the Center for Reproductive Medicine, Renmin Hospital of Wuhan University. The primary outcomes included clinical pregnancy rate of frozen-thawed embryos transfer (FET) in PPOS group, and good-quality embryo rate in both groups. The secondary outcomes were fertilization rate, egg utilization rate and cycle cancellation rate. The results showed that there were no significant differences in basal follicle stimulating hormone (bFSH), antral follicle count (AFC), duration and total dosage of gonadotropin (Gn), number of oocytes retrieved, intracytoplasmic sperm injection (ICSI) rate, fertilization rate, and cycle cancellation rate between the two groups (P>0.05). However, the oocyte utilization rate and good-quality embryo rate in PPOS group were significantly higher than those in non-PPOS group (P<0.05). By the end of April 2017,62 FET cycles were conducted in PPOS group. The clinical pregnancy rate and embryo implantation rate were 22.58% and 12.70%, respectively. In conclusion, PPOS protocol may provide better clinical outcomes by improving the oocyte utilization rate and good-quality embryo rate for aged infertile patients who failed to get pregnant in the first IVF/ICSI-ET cycles.

Dual trigger of triptorelin and HCG optimizes clinical outcome for high ovarian responder in GnRH-antagonist protocols.

In this paper, a retrospective cohort study was conducted to the high ovarian responders in GnRH-antagonist protocols of IVF/ICSI cycles. The purpose of the study is to investigate whether dual triggering of final oocyte maturation with a combination of gonadotropin-releasing hormone (GnRH) agonist and human chorionic gonadotropin (HCG) can improve the clinical outcome compared with traditional dose (10000IU) HCG trigger and low-dose (8000IU) HCG trigger for high ovarian responders in GnRH-antagonist in vitro fertilization/intracytoplasmic sperm injection (IVF-ICSI) cycles. Our study included 226 couples with high ovarian responders in GnRH-antagonist protocols of IVF/ICSI cycles. Standard dosage of HCG trigger (10000 IU of recombinant HCG) versus dual trigger (0.2 mg of triptorelin and 2000 IU of recombinant HCG) and low-dose HCG trigger (8000IU of recombinant HCG) were used for final oocyte maturation. Our main outcome measures were high quality embryo rate, the number of usable embryos, the risk of OHSS, duration of hospitalization and incidence rate of complications. Our evidence demonstrated that dual trigger is capable of preventing severe OHSS while still maintaining excellent high quality embryo rate in in high ovarian responders of GnRH-antagonist protocols.

Role of osteopontin in decidualization and pregnancy success.

OPN is essential for blastocyst implantation and placentation. Previous study found that miR181a was increased while miR181b was downregulated in endometrium during decidualization. However, the information regarding their effects on decidualization in human endometrium is still limited. Here, we report a novel role of OPN and miR181b in uterine decidualization and pregnancy success in humans. The expression of OPN was high in endometrium in secretory phase and in vitro decidualized hESC, whereas miR181b expression was low in identical conditions. Further analysis confirmed that OPN expression was upregulated by cAMP and C/EBPß signal pathway, while downregulated by miR181b. Increased OPN expression could promote the expression of decidualization-related and angiogenesis-related genes. Conversely, the processes of decidualization and angiogenesis in hESC were compromised by inhibiting OPN expression in vitro OPN expression was repressed in implantation failure group when compared with successful pregnancy group in IVF/ICSI-ET cycles. These findings add a new line of evidence supporting the fact that OPN is involved in decidualization and pregnancy success.

Patients with cervical Ureaplasma Urealyticum and Chlamydia Trachomatis infection undergoing IVF/ICSI-ET: The need for new paradigm.

Genital tract infections with ureaplasma urealyticum (UU) and chlamydia trachomatis (CT) are the most frequent sexually-transmitted disease worldwide. UU and CT infections are considered to be the leading cause for infertility and adverse pregnancy outcomes. However, little is known about the specific effect of cervical UU and CT infections on the etiology of female infertility, as well as the pregnancy outcomes of the patients undergoing in vitro fertilization/intracytoplasmic sperm injection-embryo transfer (IVF/ICSI-ET). In order to find the association between cervical UU and/or CT infection and pregnancy outcomes, we conducted a retrospective case-control study on the patients undergoing IVF/ICSI-ET with cervical UU and/or CT infection. A total of 2208 patients who received IVF/ICSI-ET were enrolled in this study. Data on the general conditions, pregnancy history and clinical pregnant outcomes were analyzed in terms of the cervical UU and CT detection. Our results revealed that cervical UU and CT infections were the risk factors for ectopic pregnancy and tubal factor-induced infertility. Moreover, the pregnancy rate, abortion rate, ectopic pregnancy rate and premature birth rate in patients with UU and/or CT infections showed no significant difference when compared with the control group. We recommend that cervical UU and CT detection should be an optional item for infertility patients and clinical UU detection should differentiate the subtypes of cervical UU. Positive cervical UU and CT infections should not be taken as strict contraindications for IVF/ICSI-ET.

Fyn Plays a Pivotal Role in Fetomaternal Tolerance Through Regulation of Th17 Cells.

PROBLEM: Spontaneous abortion is a poorly understood phenomenon, although fetomaternal intolerance is known to play an important role in its pathogenesis. The tyrosine-specific phosphotransferase Fyn has been reported as a significant regulator in immune response. However, its role in fetomaternal immune tolerance and contribution to spontaneous abortion remains unclear. METHODS OF STUDY: Fyn expression was evaluated at the fetomaternal interface of normal pregnant and abortion-prone mice, as well as in decidual tissue obtained from normal human pregnancies and idiopathic miscarriages. A Fyn inhibitor was administrated into the LPS-induced abortion mice to investigate the variation of embryo resorption and local immunity. RESULTS: Fyn expression fluctuated with the progress of normal pregnancy and was elevated in abortion-prone mice and patients with recurrent spontaneous abortion. The Fyn inhibitor reversed LPS-induced embryo absorption and aberrant inflammatory status including redundant expression of IRF4 and increased proportion of Th17 cells. CONCLUSION: Fyn is confirmed as a negative regulator in fetomaternal immune tolerance, through promoting Th17 cell expansion and proinflammatory factors expression.

Anakinra and etanercept prevent embryo loss in pregnant nonobese diabetic mice.

Bacteria and viruses activate the host innate immune response via Toll-like receptor (TLR)-involved signaling and potentially cause pregnancy failure. TLR7 and TLR9 respond to single-stranded RNA (a viral intermediate) and hypomethylated CpG DNA motifs (specific molecular constituents of bacteria) respectively. In this study, we treated murine RAW264.7 cells with R837, CpG1826, or a combination of the two. RT-PCR was performed to detect cytokines, Tlr7, and Tlr9. WT and nonobese diabetic murine embryo resorption models were established by i.p. injections of TLR7 and TLR9 ligands. Neutralizing antibodies and the IL1ß and TNFα inhibitors were used. The specific inhibitors anakinra and etanercept effectively prevented TLR7 and TLR9 ligand-induced embryo loss. Notably, this effect was not observed in decidual NK cell-depleted mice. Our findings suggest that anakinra and etanercept may have potential for preventing TLR7 or TLR9 ligand-induced abortion in the presence of decidual NK cells.

Osteopontin facilitates invasion in human trophoblastic cells via promoting matrix metalloproteinase-9 in vitro.

Successful implantation of embryo and placentation depend on proper trophoblast proliferation and differentiated into specialized invasive trophoblast. However, little is known about the regulatory factors and mechanisms in trophoblast proliferation and differentiation. Osteopontin (OPN) is a member of the small integrin-binding ligand N-linked glycoprotein family and participates in cell adhesion and invasion. It has been identified that OPN is highly expressed in invasive trophoblasts in human placenta. In this study, we demonstrated that OPN is constitutively expressed in highly invasive phenotype of human choriocarcinoma cell lines of JAR and JEG-3 cells, and OPN could promote trophoblast proliferation and invasion, partly through promoting MMP-9 secretion. Inhibition of OPN will compromise the abilities of proliferation and invasion in JAR and JEG-3 cell lines. Our data showed that the expression of OPN in trophoblast may participate in placentation, OPN expression defects may be involved in gestational trophoblastic diseases.

Osteopontin is expressed in the oviduct and promotes fertilization and preimplantation embryo development of mouse.

Osteopontin (OPN) is a multifunctional phosphoprotein that is detected in various tissues, including male and female reproductive tracts. In this study, we evaluated OPN expression in mouse oviducts during the estrus cycle, and at days 1-5 of pregnancy and pseudopregnancy by reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry. The mice oocytes, sperm and embryos were treated with different concentrations of anti-OPN antibody in vitro to detect the function of OPN in fertilization and preimplantation embryo development. OPN mRNA and protein expression in mouse oviducts were cyclic dependent throughout the estrous cycle, which was highest at estrous and lowest at diestrous. Such a phenomenon was consistent with the change in estrogen level in mice. The expression levels of OPN in mice oviduct of normal pregnancy and pseudopregnancy were significantly different, which indicated that OPN expression in mouse oviducts was depend on estrogen and preimplantation embryo. Furthermore, anti-OPN antibody treatment could reduce the rates of fertilization, cleavage and blastocyst formation in vitro in a dose-dependent way. Overall, our results indicated that the expression of OPN in mouse oviducts during the estrous cycle and early pregnancy is likely regulated by estrogen and the embryo, and OPN may play a vital role in oocyte fertilization and preimplantation embryo development.

Osteopontin is expressed in the mouse uterus during early pregnancy and promotes mouse blastocyst attachment and invasion in vitro.

Embryo implantation into the maternal uterus is a decisive step for successful mammalian pregnancy. Osteopontin (OPN) is a member of the small integrin-binding ligand N-linked glycoprotein family and participates in cell adhesion and invasion. In this study, we showed that Opn mRNA levels are up-regulated in the mouse uterus on day 4 and at the implantation sites on days 5 and 8 of pregnancy. Immunohistochemistry localized the OPN protein to the glandular epithelium on day 4 and to the decidual zone on day 8 of pregnancy. OPN mRNA and proteins are induced by in vivo and in vitro decidualization. OPN expression in the endometrial stromal cells is regulated by progesterone, a key regulator during decidualization. As a secreted protein, the protein level of OPN in the uterine cavity is enriched on day 4, and in vitro embryo culturing has indicated that OPN can facilitate blastocyst hatching and adhesion. Knockdown of OPN attenuates the adhesion and invasion of blastocysts in mouse endometrial stromal cells by suppressing the expression and enzymatic activity of matrix metalloproteinase-9 in the trophoblast. Our data indicated that OPN expression in the mouse uterus during early pregnancy is essential for blastocyst hatching and adhesion and that the knockdown of OPN in mouse endometrial stroma cells could lead to a restrained in vitro trophoblast invasion.