METTL3 is essential for normal progesterone signaling during embryo implantation via m6A-mediated translation control of progesterone receptor.

Embryo implantation, a crucial step in human reproduction, is tightly controlled by estrogen and progesterone (P4) via estrogen receptor alpha and progesterone receptor (PGR), respectively. Here, we report that N6-methyladenosine (m6A), the most abundant mRNA modification in eukaryotes, plays an essential role in embryo implantation through the maintenance of P4 signaling. Conditional deletion of methyltransferase-like 3 (Mettl3), encoding the m6A writer METTL3, in the female reproductive tract using a Cre mouse line with Pgr promoter (Pgr-Cre) resulted in complete implantation failure due to pre-implantation embryo loss and defective uterine receptivity. Moreover, the uterus of Mettl3 null mice failed to respond to artificial decidualization. We further found that Mettl3 deletion was accompanied by a marked decrease in PGR protein expression. Mechanistically, we found that Pgr mRNA is a direct target for METTL3-mediated m6A modification. A luciferase assay revealed that the m6A modification in the 5' untranslated region (5'-UTR) of Pgr mRNA enhances PGR protein translation efficiency in a YTHDF1-dependent manner. Finally, we demonstrated that METTL3 is required for human endometrial stromal cell decidualization in vitro and that the METTL3-PGR axis is conserved between mice and humans. In summary, this study provides evidence that METTL3 is essential for normal P4 signaling during embryo implantation via m6A-mediated translation control of Pgr mRNA.

Trophectoderm biopsy reduces the level of serum ß-human chorionic gonadotropin in early pregnancy.

OBJECTIVE: To assess whether trophectoderm biopsy has any impact on the level of serum ß-human chorionic gonadotropin (ß-hCG) in early pregnancies. DESIGN: Retrospective cohort study. SETTING: University-affiliated reproductive medical center. PATIENT(S): Three hundred and eighty-three women undergoing 396 frozen embryo transfer (FET) cycles with preimplantation genetic testing (PGT), and 353 women undergoing 465 FET cycles with in vitro fertilization or intracytoplasmic sperm injection, all women having positive serum ß-hCG results on the 12th day after blastocysts transfers. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Serum ß-hCG levels on the 12th day after warmed blastocyst transfer and perinatal outcomes of clinical pregnancy. RESULTS: The diagnostic threshold of serum ß-hCG levels on the 12th day after FET for prediction of a live birth was 368.55 mIU/mL with an area under the curve of 0.791 (0.729∼0.853) in the biopsy group, which was lower than the 411.45 mIU/mL in the control group. The average level of serum ß-hCG in the biopsy group with clinical pregnancies was statistically significantly lower than that of the control group: 703.10 (569.63) versus 809.20 (582.00), respectively. No statistically significant differences in perinatal outcomes, including gestational age, hypertensive disorder in pregnancy, and neonatal malformation, were found between the two groups. CONCLUSION(S): Trophectoderm biopsy may reduce the level of serum ß-hCG in early pregnancies (the 12th day after embryo transfer), but no increased risk was found of adverse perinatal outcomes after trophectoderm biopsy.

Higher chromosomal abnormality rate in blastocysts from young patients with idiopathic recurrent pregnancy loss.

OBJECTIVE: To determine whether the incidence of chromosomal abnormalities in blastocysts is higher in patients with idiopathic recurrent pregnancy loss (iRPL) who underwent preimplantation genetic testing for aneuploidy (PGT-A) than in those who underwent preimplantation genetic testing for monogenic defects (PGT-M). DESIGN: Retrospective cohort study. SETTING: University-affiliated reproductive center. PATIENT(S): A total of 62 patients with iRPL underwent 101 PGT-A cycles (iRPL group), and 212 patients underwent 311 PGT-M cycles (control group). INTERVENTIONS(S): Blastocyst biopsy and comprehensive chromosome screening technologies, including single-nucleotide polymorphism microarrays and next-generation sequencing. MAIN OUTCOME MEASURE(S): Incidence of chromosomal abnormalities in blastocysts and clinical miscarriage (CM) rate. RESULT(S): Stratification analysis by maternal age showed an increased incidence of chromosomal abnormalities in the iRPL group aged ≤35 years (48.9% vs. 36.9%), whereas no significant increase was found in the iRPL group aged >35 years (66.9% vs. 61.4%). After transfer of euploid embryos, women aged ≤35 years with iRPL exhibited an increased CM rate compared with the control group (26.1% vs. 3.1%). CONCLUSION(S): Young patients with iRPL have a significantly higher rate of chromosomal abnormalities in blastocysts compared with patients with no or sporadic CM. Although euploid embryos were transferred after PGT-A, young patients with iRPL had a higher CM rate, which may indicate that chromosomal abnormalities might not be the only causal factor for iRPL. Therefore, the role of PGT-A in iRPL still needs to be clarified.

Number of blastocysts biopsied as a predictive indicator to obtain at least one normal/balanced embryo following preimplantation genetic diagnosis with single nucleotide polymorphism microarray in translocation cases.

PURPOSE: The aim of this study is to investigate the minimum number of blastocysts for biopsy to increase the likelihood of obtaining at least one normal/balanced embryo in preimplantation genetic diagnosis (PGD) for translocation carriers. METHODS: This blinded retrospective study included 55 PGD cycles for Robertsonian translocation (RT) and 181 cycles for reciprocal translocation (rcp) to indicate when only one of the couples carried a translocation. Single-nucleotide polymorphism microarray after trophectoderm biopsy was performed. RESULTS: Reliable results were obtained for 355/379 (93.7 %) biopsied blastocysts in RT group and 986/1053 (93.6 %) in rcp group. Mean numbers of biopsied embryos per patient, normal/balanced embryos per patient, and mean normal/balanced embryo rate per patient were 7.4, 3.1, and 40.7 % in RT group and 8.0, 2.1, and 27.3 %, respectively, in rcp group. In a regression model, three factors significantly affected the number of genetically transferrable embryos: number of biopsied embryos (P = 0.001), basal FSH level (P = 0.040), and maternal age (P = 0.027). ROC analysis with a cutoff of 1.5 was calculated for the number of biopsied embryos required to obtain at least one normal/balanced embryo for RT carriers. For rcp carriers, the cutoff was 3.5. The clinical pregnancy rate per embryo transfer was 44.2 and 42.6 % in RT and rcp groups (P = 0.836). CONCLUSIONS: The minimum numbers of blastocysts to obtain at least one normal/balanced embryo for RT and rcp were 2 and 4 under the conditions of female age < 37 years with a basal FSH level < 11.4 IU/L.

[Combination of multiple displacement amplification with short tandem repeat polymorphismin preimplantation genetic diagnosis].

OBJECTIVE: To explore the application of multiple displacement amplification (MDA) combined with short tandem repeats (STRs) in preimplantation genetic diagnosis (PGD). METHODS: MDA was applied to amplify the whole genome of a single cell and to retrieve and assemble the highly heterogeneous STR loci among human population. Haplotype analytic system was established with aiming at diagnosis of the single gene diseases by selecting the STR loci located within the pathogenic genes or on both bounding sides of the pathogenic genes. At the same time, allele specific amplification, PCR-reverse dot-blotting hybridization methods and gene sequencing methods were employed for direct detection of the pathogenic genes. The STR loci located at related chromosomes were selected to carry out allele number analysis on the basis of chromosome number and structural abnormality. RESULTS: In the study, 12 PGD systems were set up including 6 different monogenic diseases (spinal muscular atrophy, Duchenne muscular dystrophy, X-linked chronic granulomatous disease, osteopetrosis, achondroplasia, X-linked severe combined immunodeficiency), Robertsonian translocations, α-thalassemia combined with Robertsonian translocation, α- and ß-double thalassemia, ß-thalassemia with HLA typing and DMD with HLA typing. Then 44 PGD cycles were performed for 35 couples with different kinds of inherited diseases, which resulted in 20 healthy liveborns (12 singletons and 4 twins) and 5 ongoing pregnancies. The clinical pregnancy rate was 47.7% (21/44) per PGD cycle. The overall diagnostic rate was 94.6% (367/388). The MDA failed in 3.6% (14/388) single blastomeres. The amplification rate of the subsequent PCR was 97.1% and the average allele drop out (ADO) rate was 12.6% (range: 0-47.5%). CONCLUSION: The application of MDA combined with STRs provided a generic PGD approach for different genetic disorders, especially for simultaneous diagnosis of two or more hereditary statuses. The method could greatly shorten the time of developing PGD system of new diseases, which broadens the indications of PGD.

Growth differential factor-9 inhibits testosterone production in mouse theca interstitial cells.

OBJECTIVE: To explore the effect of growth differential factor-9 (GDF-9) alone on cell proliferation, cell viability, steroidogenesis, and hormone-stimulated gene expression in cultured mouse theca interstitial cells. DESIGN: Basic research. SETTING: University hospital. ANIMAL(S): Immature 3- to 4-week-old SPF KM mice obtained from the Laboratory Animal Center of Sun Yat-Sen University. INTERVENTION(S): Addition of GDF-9 at different dosages to primary culture of mouse theca interstitial cells. MAIN OUTCOME MEASURE(S): Cell number, cell viability, progesterone and testosterone levels, and hormone-stimulated gene mRNA abundance. RESULT(S): Growth differential factor-9 mildly increased the number of mouse theca interstitial cells and cell viability in a dose-dependent manner and mildly inhibited the production of progesterone in mouse theca interstitial cells. Administration of GDF-9 at the dosages of 200 ng/mL and 400 ng/mL resulted in a significant decrease in the testosterone level compared with the control group by 60.42% and 68.76%, respectively. Growth differential factor-9 significantly suppressed Lhcgr mRNA by 47.36%, Cyp11a1 mRNA by 62.30%, and Cyp17a1 mRNA by 55.39%, but had only a mild effect on Star gene expression. CONCLUSION(S): Growth differential factor-9 can inhibit the production of testosterone in mouse theca interstitial cells and suppress the corresponding gene expression.

A further exploration of the impact of antinuclear antibodies on in vitro fertilization-embryo transfer outcome.

OBJECTIVE: To determine the presence of antinuclear antibodies (ANAs) in follicular fluid (FF) and embryos as to their potential impact on IVF outcome. METHODS: A total of 100 women presenting to IVF/ICSI program were recruited in this study, wherein 50 women sero-positive for ANA served as study group (ANA+ group) and 50 women seronegative for ANA served as control group (ANA- group), and patients were age-matched in the two groups. ANA level in serum and FF were examined by ELISA, and immunofluorescence assay was employed to detect IgG within embryos. RESULTS: In ANA+ group, 36 women showed positive ANA both in serum and FF on the day of ovum pick-up (OPU), wherein 34 exhibited fluorescence of IgG in embryos. All the 50 women in control group showed negative results in serum and FF ANA on OPU day and in embryo immunofluorescence. Serum ANA level positively correlated with FF ANA level. IVF outcomes were significantly poorer in ANA+ group, mainly reflecting the less number of high-quality embryos, reduced rates of pregnancy, clinical pregnancy, and implantation. CONCLUSION: These data demonstrated the existence of ANA in FF and embryos, which may exert detrimental impact on IVF outcome.

Insufficient histone-3 lysine-9 deacetylation in human oocytes matured in vitro is associated with aberrant meiosis.

OBJECTIVE: To characterize histone acetylation during meiosis in human oocytes matured in vitro or in vivo. DESIGN: Experimental study. SETTING: University reproductive medical center. PATIENT(S): Patients undergoing routine intracytoplasmic sperm injection (ICSI) cycles. INTERVENTION(S): Immature and mature oocytes were collected from patients undergoing intracytoplasmic sperm injection. MAIN OUTCOME MEASURE(S): Immunohistochemical assessment of the levels of acetylated lysine-9 of histone-3 (H3K9) and lysine-12 of histone-4 (H4K12) combined with spindle and chromosome configurations in in vitro- and in vivo-matured human oocytes. Transcript levels of histone deacetylases (HDACs) 1 and 2 were measured by single-cell quantitative polymerase chain reaction. RESULT(S): Acetylation of H3K9 and H4K12 decreased during human oocyte maturation. Residual H3K9 acetylation was found in 37.7% of oocytes matured in vitro, compared with 11.8% in oocytes matured in vivo. Abnormal metaphase spindle was more frequent in oocytes with residual histone acetylation than without (51.6% vs. 25.4%, respectively). Treatment with the HDAC inhibitor trichostatin A increased the incidence of an abnormal metaphase but had no adverse effect on maturation efficiency. Furthermore, expression of HDAC1 transcripts was significantly lower in oocytes matured in vitro versus in vivo. CONCLUSION(S): Reduced HDAC1 expression and insufficient histone deacetylation are associated with metaphase defects in human oocytes matured in vitro.

Differentially expressed micoRNAs in human oocytes.

PURPOSE: To identify differentially expressed microRNAs (miRNAs) and expression patterns of specific miRNAs during meiosis in human oocytes. MATERIALS AND METHODS: To identify differentially expressed miRNAs, GV oocytes and MII oocytes matured at conventional FSH levels (5.5 ng/ml) were analyzed by miRNA microarray. Real-time RT-PCR was used to confirm the changed miRNAs. To validate the dynamic changes of miRNAs from GV to MII stages, oocytes were divided into four groups (#1-4), corresponding to GV oocytes, MI oocytes, MII oocytes matured in conventional FSH level and MII oocytes matured in high FSH level (2,000 ng/ml) respectively. RESULTS: Compared with GV oocytes, MII oocytes exhibited up-regulation of 4 miRNAs (hsa-miR-193a-5p, hsa-miR-297, hsa-miR-625 and hsa-miR-602), and down-regulation of 11 miRNAs (hsa-miR-888*, hsa-miR-212, hsa-miR-662, hsa-miR-299-5p, hsa-miR-339-5p, hsa-miR-20a, hsa-miR-486-5p, hsa-miR-141*, hsa-miR-768-5p, hsa-miR-376a and hsa-miR-15a). RT-PCR analysis of hsa-miR-15a and hsa-miR-20a expression revealed concordant dynamic changes in oocytes from group 1 to group 4. CONCLUSION(S): Specific miRNAs in human oocytes had dynamic changes during meiosis. High-concentration FSH in IVM medium led to reverse effect on the expression of hsa-miR-15a and hsa-miR-20a.

A novel disintegrin, jerdonatin, inhibits platelet aggregation and sperm-egg binding.

A novel disintegrin, jerdonatin, was purified to homogeneity from Trimeresurus jerdonii venom by gel filtration and reversed-phase high-pressure liquid chromatography. We isolated the cDNA encoding jerdonatin from the snake venom gland. Jerdonatin cDNA precursor encoded pre-peptide, metalloprotease and disintegrin domain. Jerdonatin is composed of 72 amino acid residues including 12 cysteines and the tripeptide sequence Arg-Gly-Asp (RGD), a well-known characteristic of the disintegrin family. Molecular mass of jerdonatin was determined to be 8011 Da by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS). Jerdonatin inhibited ADP- and collagen-induced human platelet aggregation with IC50 of 123 and 135 nM, respectively. We also investigated the effect of jerdonatin on the binding of B6D2F1 hybrid mice spermatozoa to mice zona-free eggs and their subsequent fusion. Jerdonatin significantly inhibited sperm-egg binding in a concentration-dependent manner, but had no effect on the fusion of sperm-egg. These results indicate that integrins on the egg play a role in mammalian fertilization.