The Mechanism Of Human Sperm Devoid Of Germinal ACE Induced Total Fertilization Failure And Lower Fertilization Rates By Conventional IVF

Since the world’s first test-tube baby was born in1978, conventional in vitro fertilization and embryo transfer (IVF-ET) has developed for nearly40years. And this technique has treated countless infertility couples. Although improvement of clinical ovulation drugs and ovulation induction programs, as well as the commercialization of laboratory reagents, its success rate also increased, but total fertilization failure and low fertilization rates were always encountered in clinical. It also gave patients a tremendous spiritual and economic pressure.Total fertilization failure by conventional FVF means within a certain period of time after in vitro fertilization, sperm is not able to fuse with ovum to form a zygote, and in morphology, the phenomenon of2pronucleuses was not seen. The true incidence of total fertilization failure (TFF) and low fertilization (defined as<25%fertilization, LFR) occurs in5%-15%, and20%, respectively, in most conventional IVF programs with a recurrence rate of about30%-67%. Therefore, diagnosing the causes of fertilization failure in IVF is important. However, a convincing test is not yet available that can predict the risk of fertilization failure.The major causes of TFF or LFR with conventional IVF appear to be defective sperm-zona pellucida binding and penetration, which are mainly due to abnormalities of the sperm not the oocytes. Experimental results obtained from gene-manipulated animals suggest that many factors thought to be highly important for sperm-zona pellucida binding and penetration turned out to be non-essential (i.e., PH20, CD46, and acrosin). However, factors that had never been considered to be involved in fertilization were found to be essential. Seven different gene-disrupted mouse lines (Clgn, Calr3, Adam1a, Adam2, Adam3, IZUMO and gACE) all have deficiencies in sperm-ZP interactions. CLGN, CALR3, and ADAM1a are testicular proteins and are not carried over to sperm. IZUMO, expressed on the sperm inneracrosomal membrane after the acrosome reaction, was found through immunocytochemistry to be the same in all of the patients of fertilization failureMany gene disruption experiments have demonstrated that a single phenotype is attributable to the defect in the expression of ADAM3on the sperm membrane. In humans, Adam1a and Adam3genes were determined to be non-functional pseudogenes and thus were thus unable to produce functional proteins. Therefore, Adam2(also known as PH30β or fertilin β) may play an important role in human sperm.Angiotensin-converting enzyme (ACE) is a ubiquitous membrane ectoprotein found in mammalian tissues. Two isoenzymes of ACE exist in human tissues:one is somatic ACE (sACE), which is expressed in the vascular endothelial cells and epithelial cells in the proximal tubules of the kidney, brain, intestinal brush border and epididymis, while the second is gACE, expressed exclusively in developing spermatids and mature spermatozoa and is involved in sperm-egg binding. Earlier studies also indicated that gACE mRNA was first detected in late-pachytene spermatocytes, whereas gACE protein appeared only in elongating spermatids; RT-PCR and ISH analyses had revealed the persistence of ACE transcripts in human sperm nuclei.The functions of the ACE isozymes in male reproduction were investigated using knockout experiments. ACE was important for achieving IVF and the spermatozoa from mice lacking both of the ACE isozymes showed defects in transport within the oviducts and in binding to the zona pellucida. Germinal ACE knockout in mice did not influence sperm number, morphology or motility, but it did cause a defect in sperm binding to the zona pellucida of the oocyte. And this phenotype resembles some cases of human TFF.In current study, clinical characteristic of TFF group, LFR group and NFR (normal fertilization rates) gourp were first analyzed. Then we measured the mRNA and protein expression of gACE and ADAM2in three groups. Meanwhile, we screened all exons and promoter of gACE for potential mutations. And we analyzed SNP found with clinical data. Finally, we detected whether SNP influenced the transcription of gACE or not. The aim of this study is to investigate the causation of fertilization failure, and to identify a new predictor for fertilization failure, or for therapy. Part I:Human sperm devoid of germinal ACE induced total fertilization failure and lower fertilization rates by conventional IVFObjective:To analysis clinical characteristics of infertile couples with total fertilization failure and lower fertilization rates by conventional IVF, and detect gACE and ADAM2expression on sperm of male patients with fertilization failure.Methods:1. After the completion of IVF, collect sperm samples from the patients enrolled in the assisted reproduction program at Women’s Hospital, School of Medicine, Zhejiang University, China and store themat-80℃.(a)38years of age or younger and (b) more than3retrieved matured oocytes (MⅡ). Any immature, deformed or post-matured oocytes, or oocytes with certain types of abnormalities were excluded from this study. Other inclusion criteria were semen characteristics of>20%progressively motility and>20×106total sperm.2. Testicular biopsies of azoospermic men with normal spermatogenesis (OAZ, n=10), with maturation arrest at the spermatocyte stage (MA, n=10), and Sertoli cell-only syndrome (SCOS, n=10).3. Sperm samples from the individuals were divided into three groups based on fertilization rates, the TFF group (40patients) with fertilization failure of all of the available oocytes; the LFR group (50patients) with fertilization rates lower than25%; and the NFR group (50patients) with fertilization rates above60%.4. Clinical characteristics for the IVF cycle were analysised, such as age, infertility duration and number of retrieved MⅡ oocytes, fertilization rates, conventional parameters of semen quality, and reasons of infertility.5. gACE and ADAM2expression in the testis of patients with SCOS, MA, OAZ, and in sperm were detected by western blotting. 6. gACE and ADAM2expression in the sperm of TFF group, LFR goup and NFR group were detected by western blotting.7. gACE expression in the sperm of TFF group, LFR goup and NFR group were detected by indirect immunofluorescence.8. gACE and ADAM2expression in the sperm of TFF group, LFR goup and NFR group were detected by RT-qPCR.9. gACE expression was detected before capacitation, after capacitation and after induction of the acrosome reaction by A23157.Results:1. No statistically difference as regards age, infertility duration and number of retrieved MⅡ oocytes was noted among the three groups.2. When the conventional parameters of semen quality were compared, a significant difference was detected. Sperm concentration and progressive motility before sperm preparation were higher in the NFR group than in the TFF group and LFR group. But there was nonsignificant difference between the TFF and LFR group3. The rate of primary infertility and unexplained infertility in TFF group and LFR group were higher than that in NFR group.4. sACE was found in the testis of patients with Sertoli Cell-Only syndrome, maturation arrest, and normal spermatogenesis, but was not observed in sperm.5. gACE was found in the testes containing normal spermatogenesis and sperm, not in the testis of patients with Sertoli Cell-Only syndrome and maturation arrest.6. Indirect immunofluorescence of viable spermatozoa showed that gACE was localized on the post-acrosomal area, neck and midpiece of morphologically normal cells.7. Western blot analysis demonstrated that statistically differences were detected in relative gACE protein expression by western blot analysis among the three groups. Compared with the NFR group, relative gACE protein expression was reduced in the TFF group, and the LFR group respectively.8. The protein expression of gACE was significantly down-regulated by approximately fourfold in37.5%(15/40) of the TFF patients and20%(10/50) of the LFR patients. 9. The protein expression of gACE was significantly down-regulated by approximately fourfold in53.8%(15/28) of the TFF primary patients and45.5%(10/22)of the LFR primary patients.10. The protein expression of ADAM2was nonsignificant difference among TFF, LFR group and NFR group.11. gACE expression was not changed after capacitation or after induction of the acrosome reaction by A23157.Conclusions:1. TFF and LFR by converntional IVF were related to Sperm concentration and progressive motility.2. Patients with primary infertility and unexplained infertility underwent conventional IVF have a high risk for TFF and LFR.3. Normal expression of gACE on sperm is a key factor for successful fertilization Part II The mechanism of human sperm devoid of germinal ACE induced total fertilization failure and lower fertilization rates by conventional IVFObjective:To screen specific regulatory region and all exons of gACE for potential mutations, and to detect the correlation between SNPs and failed fertilization by IVF, and finally verify the effects of SNPs on transcription of gACE.Methods:1. After the completion of IVF, collect sperm samples from the patients enrolled in the assisted reproduction program at Women’s Hospital, School of Medicine, Zhejiang University, China and store themat-80℃.(a)38years of age or younger and (b) more than3retrieved matured oocytes (MⅡ). Any immature, deformed or post-matured oocytes, or oocytes with certain types of abnormalities were excluded from this study. Other inclusion criteria were semen characteristics of>20%progressively motility and>20×106total sperm.2. Sperm samples from the individuals were divided into three groups based on fertilization rates, the TFF group (40patients) with fertilization failure of all of the available oocytes; the LFR group (50patients) with fertilization rates lower than25%; and the NFR group (50patients) with fertilization rates above60%.3. Specific regulatory region was screened by PCR amplification and sequencing for potential mutation.4. The distribution of SNP in male patients by conventional IVF was detected using PCR-RFLP and the correlation of SNP and failed fertilization was analyzed.5. The effects of SNPs on transcription of gACE were verified by dual luciferase reporter assay system.6. All exons of gACE were screened by PCR amplification and sequencing for potential mutation. Results:1.23out of25patients devoid of gACE on their sperm (15patients in the TFF group and8patients in the LFR group) presented TT genotype of SNP rs4316and aa genotype of SNP rs4320.2. The frequency of the a allele of rs4320is in linkage disequilibrium with the T allele of rs4316.3. Nonsignificant differences were detected in the genotype frequency among the three groups, but compared with the NFR group, TT genotype frequency increased in the combined TFF and LFR groups.4. The T allelic frequency in the LFR group was markedly higher compared with the NFR group.5. The relative luciferase activity of pGL3-promoter-T was lower compared with pGL3-promoter-C.6. Only one patient whose sperm had normal gACE expression in the TFF group demonstrated a heterozygous substitution (pSer1005Cys).Conclusions:1. T allelic frequency of SNP rs4316and a allelic frequency of SNP rs4320were related with failed fertilization.2. TT genotype of SNP rs4316and aa genotype of SNP rs4320were related with decreased expression of gACE.