Effects of N-acetylcysteine on growth,viability and reactive oxygen species levels in small antral follicles cultured in vitro

Objective:To investigate the effects of different concentrations of N-acetylcysteine on follicular growth and morphology,as well as on viability,levels of reactive oxygen species(ROS)and meiotic progression of oocytes from in vitro cultured bovine early antral follicles.Methods:Isolated early antral follicles(about 500μm)were cultured in TCM-199+alone or supplemented with 1.0,5.0 or 25.0 mM N-acetylcysteine at 38.5℃with 5%CO_(2) for 8 days.Follicle diameters were evaluated at day 0,4 and 8 of culture.At the end of culture,the levels of ROS,chromatin configuration and viability(calcein-AM and ethidium homodimer-1 staining)were investigated in the cumulus-oocyte complexes.Comparisons of follicle diameters between treatments were performed.Data on percentages of morphologically normal follicles,growth rates and chromatin configuration in different treatments were compared.Results:An increase in follicular diameters after culture in all treatments was observed,except for follicles cultured with 25.0 mM N-acetylcysteine.Fluorescence microscopy showed that oocytes cultured in all treatments were stained positively with calcein AM,and that 5.0 mM N-acetylcysteine reduced fluorescence for ethidium homodimer-1.Intracellular levels of ROS in oocytes from follicles cultured with 1.0 mM N-acetylcysteine showed a significant reduction compared to other treatments.The presence of N-acetylcysteine in culture medium did not influence the rates of oocyte at the germinal vesicle stage.Conclusions:N-acetylcysteine at concentrations of 1.0 and 5.0 mM reduces ROS levels and staining for ethidium homodimer-1 in in vitro cultured follicles,respectively,while 25.0 mM N-acetylcysteine decreases follicular growth and the percentages of continuously growing follicles.

Anti-Müllerian hormone and antral follicle count predict ovarian response in women less than 45 years following GnRH antagonist multiple-dose protocol

Objective:To speculate which of the following parameters:antral follicle count(AFC),anti-Müllerian hormone(AMH),follicle-stimulating hormone(FSH)and age can be used as a predictor of ovarian response to gonadotropin-releasing hormone(GnRH)antagonist stimulation multiple-dose protocol in women under 45 years,and to determine the cutoff value of these parameters and their correlations for predicting low and high ovarian response.Methods:This prospective study included 462 women with the mean age of(29.3±6.5)years.All women were subjected to the GnRH antagonist stimulation multiple-dose protocol.On the second day of the menstrual cycle,ultrasonography was conducted to determine AFC in both ovaries.Peripheral blood samples were collected to evaluate the level of estradiol,FSH,luteinizing hormone,prolactin,thyroid-stimulating hormone,and AMH.The women were divided into three groups:low response(AHH<1 ng/mL,n=173),normal response(AMH=1.0-3.5 ng/mL,n=175),and high response(AMH>3.5 ng/mL,n=114).Results:A significant decrease was found in the age and FSH level in the high response group compared to other groups(P<0.001).Conversely,a significant increase was shown in AMH,estradiol on human chorionic gonadotropin(hCG)day,AFC,mature oocytes,fertilized oocytes,and embryos transferred in the high response group compared to the other two groups(P<0.001).The receiver operating characteristic(ROC)curves demonstrated that AFC and AMH had the highest accuracy,followed by basal FSH level and age in the prediction of low ovarian reserves(P<0.001)with cutoff values of≤4.50 and≤0.95 for AFC and AMH,respectively.Moreover,the ROC analysis showed that AFC had the highest accuracy,followed by AMH level and age in the prediction of high ovarian reserves with a cutoff value of≥14.50,≥3.63,and≤27.50 years,respectively(P<0.01).A significant decrease was observed in women's age,estradiol level,and oocyte fertilization rate in pregnant women compared to non-pregnant women(P<0.001).Additionally,significant negative correlations were found between the AFC,the number of mature oocytes,fertilized oocytes,embryos transferred,and the age of pregnant women(P<0.001).Conclusions:AFC and AMH predict low and high ovarian response to GnRH antagonist stimulation multiple-dose protocol in women under 45 years.

Fine mapping of genomic regions associated with female fertility in Nellore beef cattle based on sequence variants from segregating sires

Background:Impaired fertility in cattle limits the efficiency of livestock production systems.Unraveling the genetic architecture of fertility traits would facilitate their improvement by selection.In this study,we characterized SNP chip haplotypes at QTL blocks then used whole-genome sequencing to fine map genomic regions associated with reproduction in a population of Nellore(Bos indicus)heifers.Methods:The dataset comprised of 1337 heifers genotyped using a GeneSeek®Genomic Profiler panel(74677 SNPs),representing the daughters from 78 sires.After performing marker quality control,64800 SNPs were retained.Haplotypes carried by each sire at six previously identified QTL on BTAs 5,14 and 18 for heifer pregnancy and BTAs 8,11 and 22 for antral follicle count were constructed using findhap software.The significance of the contrasts between the effects of every two paternally-inherited haplotype alleles were used to identify sires that were heterozygous at each QTL.Whole-genome sequencing data localized to the haplotypes from six sires and 20 other ancestors were used to identify sequence variants that were concordant with the haplotype contrasts.Enrichment analyses were applied to these variants using KEGG and MeSH libraries.Results:A total of six(BTA 5),six(BTA 14)and five(BTA 18)sires were heterozygous for heifer pregnancy QTL whereas six(BTA 8),fourteen(BTA 11),and five(BTA 22)sires were heterozygous for number of antral follicles’QTL.Due to inadequate representation of many haplotype alleles in the sequenced animals,fine mapping analysis could only be reliably performed for the QTL on BTA 5 and 14,which had 641 and 3733 concordant candidate sequence variants,respectively.The KEGG“Circadian rhythm”and“Neurotrophin signaling pathway”were significantly associated with the genes in the QTL on BTA 5 whereas 32 MeSH terms were associated with the QTL on BTA 14.Among the concordant sequence variants,0.2%and 0.3%were classified as missense variants for BTAs 5 and 14,respectively,highlighting the genes MTERF2,RTMB,ENSBTAG00000037306(miRNA),ENSBTAG00000040351,PRKDC,and RGS20.The potential causal mutations found in the present study were associated with biological processes such as oocyte maturation,embryo development,placenta development and response to reproductive hormones.Conclusions:The identification of heterozygous sires by positionally phasing SNP chip data and contrasting haplotype effects for previously detected QTL can be used for fine mapping to identify potential causal mutations and candidate genes.Genomic variants on genes MTERF2,RTBC,miRNA ENSBTAG00000037306,ENSBTAG00000040351,PRKDC,and RGS20,which are known to have influence on reproductive biological processes,were detected.

Rejuvenation of Ovary and Thin Endometrium by Autologous PRP Injection in POR and Recurrent Implantation Failure

In India, the problem of infertility is growing and in the last 5 years, it has gone up to 20% - 30%. This ongoing prospective clinical study brings forth a novel, innovative, effective, simple, affordable, easily performed outpatient procedure (OP) and a promising therapeutic method in rejuvenating the Ageing Ovaries and Thin Endometrium, with autologous Platelet Rich Plasma (PRP). This clinical study proves to give a better result in rejuvenating Ovary and treating the Thin Endometrium. This pilot study included five women (28 - 44 years) with Poor Ovarian Response (POR), Premature Ovarian Insufficiency (POI) and Perimenopause and Thirty-nine women (22 - 43 years.) with recurrent implantation failure due to Thin Endometrium were subjected to autologous PRP instillation under Ultrasound Guidance, and Hysteroscopic guided PRP. After PRP, a significant output was obtained, with improved Anti Mullerian Hormone (AMH) and Antral Follicle Count (AFC) and out of five women three women conceived by Intra Cytoplasmic Sperm Injection (ICSI). PRP injected in women with Poor Ovarian Response found successful ovarian rejuvenation within 1 - 3 months and had a 60% of pregnancy rate, PRP into the endometrium had 53.8% successful pregnancies. We have not encountered any complications.

Anti-Müllerian hormone, antral follicle count and folliclestimulating hormone for predicting the number of oocytes retrieved in IVF/ICSI cycles

Objective To evaluate the efficacy of anti-Müllerian hormone（AMH）, antral follicle count（AFC） and follicle-stimulating hormone（FSH） for predicting the number of oocytes retrieved in in-vitro fertilization/intracytoplasmic sperm injection（IVF/ICSI）cycles.Methods In this retrospective study, a total of 122 infertile women were divided into two groups： group A, 〈35 years（n=71）; group B, ≥35 years（n=51）. AMH and FSH were determined on 2-5 d of the early menstrual cycle. AFC was tested on the second day of the menstrual cycle before the start of stimulation.Results Group B had higher FSH levels compared with group A（8.2±3.5 IU/L vs 6.8±2.4 IU/L, P〈0.05）. However, levels of AMH and AFC in group B were lower than those of group A（AMH： 4.2±3.5 μg/L vs 2.7±2.7 μg/L; AFC： 9.0±3.9 vs 5.4±3.3, P〈0.05）. The number of oocyte retrieved in the two groups was not significantly different（11.5±6.8 vs 9.6±6.9, P〉0.05）. The level of AMH was more strongly correlated with the number of oocytes retrieved than that of AFC or FSH level. The strengths of the correlation degrees were AMH level, AFC, and FSH level in turn（r=0.600, 0.511,-0.369）.Conclusion AMH would be a useful predictor for ovarian response.

Expression of Cellular FLICE-like Inhibitory Protein in Granulosa Cells in In Vitro Fertilization Patients with Advanced Endometriosis

Background:This study evaluated the expression of cellular FLICE-like inhibitory protein(cFLIP)in granulosa cells(GCs)obtained from in vitro fertilization-embryo transfer patients with advanced endometriosis.Methods:A total of 267 patients with advanced endometriosis were enrolled in this study.They were divided into clinical pregnancy group(n=114)and nonpregnancy group(n=153).The expressions of cFLIP in mRNA and protein level were measured by real-time polymerase chain reaction(RT-PCR)and Western blotting.The related factors on the clinical pregnancy were analyzed using logistic regression analysis.Coefficients of correlation were calculated using the nonparametric rho-Spearman test.Results:The number of oocytes retrieved,fertilization rate,and cleavage rate were significantly and independently related with clinical pregnancy(P>0.05).RT-PCR and Western blotting analysis showed that the expressions of cFLP in mRNA and protein level were significantly higher in the clinical pregnancy group than in nonpregnancy group(P>0.05).cFLIP had a significantly positive correlation with the number of oocytes retrieved(P>0.05)and no correlation with fertilization rate and cleavage rate(P<0.05).Conclusion:Higher expression of cFLIP increased the pregnancy rate in women with advanced endometriosis.