Analysis Of Factors Affecting Clinical Pregnancy Rate By Artificial Insemination With Donor Semen And Molecular Biomarkers In Human Seminal Plasma Of Patients With Non-obstructive Azoospermia

Objective:This study aimed to explore the multiple influence factors affecting the clinical pregnancy rate by artificial insemination with donor semen(AID)and to investigate the expression profiles of miRNA in seminal plasma of patients with different pathological patterns of non-obstructive azoospermia(NOA)to explore the potential molecular biomarkers which can provide the clue for the diagnosis and the treatment of NOA.Methods:1.Totally 3880 AID cycles of 2030 infertile couples who sought for diagnosis and treatment from January 2010 to December 2016 in Technical Service Center of Family Planning and Reproductive Health were retrospectively analyzed.The factors affecting clinical pregnancy outcomes of AID including age,infertile period,infertile type of the infertile women,number of AID cycles,treatment protocols,AID operative approaches,semen parameters,and etc.Statistical analysis was conducted on these factors through single-factor analysis and Logistic regression analysis.2.The miRNA expression profiles in human seminal plasma of 13 NOA patients in our center with different pathological patterns and 3 fertile controls were performed by high-throughput sequencing technology.3.A stem-loop quantitative reverse-transcription PCR(qRT-PCR)assay was conducted to confirm the concentrations of the miR-34c-5p and miR-202-3p in 10 patients with SCOS,10 patients with SA,and 10 fertile controls.4.qRT-PCR assay was conducted to confirm the concentrations of the target gene Hes5 in 5 patients with SCOS,5 patients with SA,and 5 fertile controls.Results:Of the 2030 AID patients,915 were clinically pregnant.The clinical pregnancy rate was 45.1%,the cycle clinical pregnancy rate was 23.58%.1.Analysis of factors affecting the clinical pregnancy rate by AID:(1)Age and pregnancy rate were negatively correlated.(2)The pregnancy probability of secondary infertile women was significantly higher than that of primary infertile women.(3)The pregnancy rate of one-time intracervical insemination(ICI)was the lowest among 4 operative approaches.(4)Ovulation induction could not increase the pregnancy rate of AID.but with an increased risk of multiple pregnancies.(5)No statistically significant relationship between pregnancy rate and the number of AID cycles,the infertile period,the progressive motile sperm count,or the sperm concentration was found.2.Analysis of the molecular biomarkers in human seminal plasma of patients with NO A:(1)miRNA expression profiles in human seminal plasma of NOA patients with different pathologic patterns:?A total of 3170 miRNAs were predicted,among which 1667 were known miRNAs and 1440 were novel miRNAs.? A total of 78 miRNAs were found to be differently expressed in patients with sertoli cell only syndrome(SCOS)compared with controls,with 4 up-regulated and 74 down-regulated miRNAs.Thirty-five miRNAs were found to be differently expressed in patients with spermatogenesis arrest(SA)compared with controls,with 3 up-regulated and 32 down-regulated miRNAs.Nine miRNAs were found to be differently expressed in SCOS patients compared with SA patients,all differentially expressed miRNAs were up-regulated.? Based on these differentially expressed miRNAs,trees with clear distinction among SCOS group,SA group,and control group were generated by Cluster analysis.? All miRNAs predicted 19,669 targeted genes,and 19,651 targeted genes were annotated.The gene ontology(GO)and KEGG pathway analysis were conducted for these annotated targeted genes.The most significant pathway that was identified by KEGG analysis was the "NOTCH signaling pathway".(2)qRT-PCR results were consistent with the miRNAs microarray results,revealed that miR-34c-5p and miR-202-3p were significantly decreased in seminal plasma of NOA patients compared with fertile controls(P<0.05).The concentration of the target gene Hes5 in SA group was significantly higher than that of fertile group(P<0.05).Conclusions:AID is a safe and effective assisted reproductive technique;Age is the most important factor affecting the clinical pregnancy rate of AID;The infertile type and the operateion method are important factors affecting the clinical pregnancy rate of AID;Ovulation induction could not increase the pregnancy rate of AID but with an increased risk of multiple pregnancy;There are no correlation of the PTMS and the concentration of sperm with the clinical pregnancy rate of AID.The conventional sperm parameters could not predict the AID pregnancy results,thus it is imperative to construct a more effective sperm functional appraisal system.The expression profiles of miRNAs in seminal plasma are altered in patients with different pathological patterns of NOA.Many target genes of the different miRNAs are enriched on the NOTCH signaling pathway.The expression abundance of miR-34c-5p and miR-202-3p in seminal plasma of patients with NOA in different pathologic patterns are significantly different.The testis specific miR-34c-5p and miR-202-3p in seminal plasma are potential biomarkers for estimating the male fertility.It is useful for understanding the molecular mechanism of NOA by exploring the effects of miR-34c-5p and miR-202-3p in seminal plasma,which can provide methods for convenient and efficient diagnosis and therapy for NOA.