Effects And The Potential Mechanisms Of IMSI On Intracytoplasmic Sperm Injection Cycles Outcomes

BACKGROUNDWith the development of our society, there are more and more couples encounter infertility, there couples,a part of them due to female factors, statistical data illustrate the male factors has risen to 50 percent with upward tending, males with abnormal semen qualities becoming a very important factors of infertility. Since 1992, ICSI (intracytoplasmic sperm injection) was introduced to address the treatment of male infertility and succeeded, there are more and more infertile couples achieve pregnancy. These are the indication of ICSI:1, abnormal semen parameters;2,Two or more times of unsuccessful trial of IVF-ET or the fertilization rate<30%.3,in vitro maturation if unstimulatedimmature oocytes systems, patients with PGD.4, patients with problem during ejaculation.5,obstructive azoospermia caused by different reasons.6, non-obstructive azoospermia ect. The implantation rate reached 70%, improve the implantation rate for the patients with poor semen parameters largely.,becoming the second revolution of the development history of assisted reproductive technology, resolve reproductive problems for more and more infertile men. There is a closed relationship between sperm morphology and functions, the morphology of sperm is regard as the predictive indicators for nature conception、IUK IVF. In the traditional ICSI project,it is usually performed under an overall optical magnification of 400×, this method shows severe limitations, which can only observe the major sperm morphological defects, minor defects that seem to be related to poor ICSI outcomes are often overlooked, it bypasses the process of natural selection and increased incidence of chromosomal abnormalities of paternal origin in children which most likely the cause of failed of ICSI cycles.Since 2001 Bartoov described motile sperm organelle morphology examination(MSOME) and then combined it with the microinjection of spermatozoa, which was called intracytoplasmic morphologically selected sperm injection (IMSI), it became the most studied one, which uses differential interferential contrast microscopy and high magnification (>x6300) enables better assessment of a spermatozoon’s morphology (such as head, neck, tail and acrosome etc.) and visualization of sperm head vacuoles which are not visible (particularly when they are small) at a conventional ICSI magnification (magnification of x200-×400).For the indications of IMSI, there are many studies reported that for the patients with several failure cycles, using IMSI can improve the implantation, clinical pregnancy rate, reduce abortion rate. Except several failure cycles of ICSI, there studies indicate that IMSI can improve the implantation and clinical pregnancy rates.There study used the IMSI for the globozoospermia patients, selected the normally morphological sperm and injected into oocytes, the wife of the patients get pregnant at last.There studies address that:IMSI can improve the blastocyst rate. However, there also some studies report estimate IMSI of no help to improve the outcomes of ICSI cycles for infertility patients. Meanwhile, the relationship between morphlogy and the state of genetic maternal of sperms becoming the focus of many research, most popular is the relationship between big head vacuole and sperm DNA fragment rate; however, Chromatin compaction abnormalities, Sperm chromosome abnormalities may also to morphology of sperm, but questions of whether morphology relate to genetic maternal and whether impact on the outcomes of ICSI cycles need further research. Therefore, this study aim to investigate whether IMSI can improve the outcomes of patients with male infertility, if it could, what is the appropriate potential indicator of IMSI and by what mechanism does IMSI improve the outcomes, whether through selecting normality morphological sperm. There study used the IMSI for the globozoospermia patients, selected the normally morphological sperm and injected into oocytes, the wife of the patients get pregnant at last.There studies address that:IMSI can improve the blastocyst rate. However, there also some studies report estimate IMSI of no help to improve the outcomes of ICSI cycles for infertility patients. Meanwhile, the relationship between morphlogy and the state of genetic maternal of sperms becoming the focus of many research, most popular is the relationship between big head vacuole and sperm DNA fragment rate; however, Chromatin compaction abnormalities* Sperm chromosome abnormalities may also to morphology of sperm, but questions of whether morphology relate to genetic maternal and whether impact on the outcomes of ICSI cycles need further research. Therefore, this study aim to investigate whether IMSI can improve the outcomes of patients with male infertility, if it could, what is the appropriate potential indicator of IMSI and by what mechanism does IMSI improve the outcomes, whether through selecting normality morphological sperm.Objectives1. We use motile sperm organellar morphology examination during cycles of intracytoplasmic sperm injection to test whether IMSI(intracytoplasmic injection of morphologically selected spermatozoa) can improve the biological and clinical outcomes in patients with male factor infertility over traditional ICSI protocols.2. We use MSOME (motile sperm organellar morphology examination systems), spermatozoa morphology may be assessed directly on motile spermatozoa at high magnification (*6600). The aim of our study was to define some MSOME parameters to establish a potential relationship between conventional semen samples and ICSI (intracytoplasmic sperm injection) outcomes,finally to find a most discriminative MSOME parameter and to investigate the influence of the MSOME parameter on the biochemical pregnancies outcomes in couples undergoing intracytoplasmic sperm injection.3. to determine the incidence of abnormal chromosomal karyotypes in the morphologically different sperms assessed by MSOME under×6600 high magnification from severe oligoasthenoteratozoospermia patients and to evaluate the influence of the magnification system on pregnancy outcome.Method1. we performed IMSI for 82 patients diagnosed with obstructive azoospermia at high magnification (×6600)and traditional ICSI (intracytoplasmic sperm injection) for the other 91 patients using testicular sperm.we also performed IMSI for 44 patients with teratozoospermia at high magnification (×6600)and traditional ICSIfor another 91 patients using ejaculated sperm.we comparatively analyzed the biological and clinical outcomes between the two techniques.2. A total of 50 semen samples were obtain from males diagnosed as oligoasthenoterazoospermia and undergoing ICSI in Center for Reproductive Medicine, Chengdu Jingjiang Hospital, every semen sample was divided into two,one of theme was undergoing conventional semen analysis,the other was firstly observed at low magnification(×400),we selected 30 normal sperms,then we use MSOME system to assessed them at high magnification,the MSOME parameters(the rate of sperm with normal morphology and the rate of sperm with big head vacuolization which relative area to sperm head>4%) were recorded.Sperman correlation analysis was used to identified the relationship between MSOME parameters and conventional semen parameters,male age, ICSI fertilization rate,cleavage rate,D3 top quality embryo rate,blastocyst formation rate.The effect of female age,infertility duration,MSOME and sperm parameters on biochemical pregnancies outcomes of ICSI cycles was analyzed by Logistic regression.ROC curve analysis was estimated to assess the prognostic ability of Logistic regression on biochemical pregnancies outcomes. If there was one MSOME parameter had significant effect on biochemical pregnancies outcome in the Logistic regression analysis,to examine the performance of MSOME parameter to predict a biochemical pregnancies outcome,ROC curve was constructed and an optimized threshold was determined,the discriminative performance of the model was assessed by the area under ROC curve.3. Three infertile patients with severe oligoasthenoteratozoospermia undergoing ICSI in Center for Reproductive Medicine, Chengdu Jingjiang Hospital, were enrolled in this study from March to December 2015, ejaculates from them were divided into two, one of them were evaluate were evaluated for sperm parameters, the rest were analyzed by MSOME system under High-magnification microscopy and then selected five morphological normal sperms and ten sperms with vacuoles(a vacuole area>4% of the head area) for each sample. Each sperm cells were under a new method of WGA (whole-genome amplification) to generate enough DNA for sequencing. We detected digitized copy-number variations (CNVs) of each sperm cell, these results allow us to identify the chromosomal karyotypes and to detect the aneuploidy of each single sperm cell.Result1. In the patients with obstructive azoospermia,there were no statistically significant difference in rates of cleavage (95.5% vs 96.7%), D3 top quality embryo (28.2% vs 29.2), implantation(26.4% vs 32.3%), pregnancy (47.3% vs 50%), blastocyst formation (54.3% vs 54.6%), abortion(14% vs 7.3%) (P>0.05), A significantly higher normal fertilization rate(77% vs 84.3%) was achieved in the IMSI group compared with the ICSI group (P＜0.05). In the patients with teratozoospermia,there were no statistically significant difference in rates of cleavage (96.2% vs 95.2%), D3 top quality embryo (27.6% vs 27.1), implantation(28.2% vs 30.7%), pregnancy (43.7% vs 43.2%),abortion(9.7% vs 10.5%) (P>0.05). the normal fertilization rate (68% vs 75.5%) and the blastocyst formation rate(54.6% vs 67.9%)was significantly higher in the IMSI group compared with the ICSI group (P<0.05).2. There were significant correlation between the two MSOME parameters and percentage of normal forms and the blastocyst formation rate (p<0.05).MSOME parameter (the rate of sperm with normal morphology) has the positive correction with percentage of normal forms and the blastocyst formation rate (R=0.670, P=0.000; R=0.513, P=0.000);another MSOME parameter(the rate of sperm with big head vacuolization which relative area to sperm head >4%) has the negative correction with percentage of normal forms and the blastocyst formation rate (R=-0.566, P=0.000; R=-0.356, P=0.003,they have no significant correction with other factors. The MSOME parameter(the rate of sperm with big head vacuolization which relative area to sperm head>4%),progressive sperm motility and progressive a+b combined motility had a significant effect on biochemical pregnancies outcome (p＜0.05).Logistic regression had predictive value for biochemical pregnancies outcome (the area under ROC curve:0.890; sensitivity:90.91%; specificity:84.6%).The AUCROC obtained with MSOME parameter(the rate of sperm with big head vacuolization which relative area to sperm head>4%) was 0.828 and a percentage of 56.2 of he rate of sperm with big head vacuolization which relative area to sperm head>4% was assumed as a cut off to evaluate biochemical pregnancies outcome(sensitivity:86.96%; specificity:69.23%),the study population was divided into two groups(A and B) according to the cut off value,there was a significant difference of cleavage rate (98.0% vs 91.5%).biochemical pregnancies outcome rate (61.8% vs 7.1%), implantation rates (81.0% vs 0) between the two groups (p<0.05),had no difference in other factors (p>0.05)3. we detect the aneuploidies and other structural abnormalities of all chromosomes of 36 sperms, there are 24 vacuole sperms and 12 normal morphology sperms, in the vacuole group there are 14 with abnormal molecular karyotype,12 sperms Loss or gain of single entire autosomes,4 of them with Sex chromosome disomy,1 of them Loss or gain of part of chromosome, in the normal group there are 5 sperms with abnormal molecular karyotype,5 sperms Loss or gain of single entire autosomes,1 of them with Sex chromosome disomy, none of them Loss or gain of part of chromosome. The rate of abnormal molecular karyotype of the two groups are(58.3% vs 41.7%), rate of sperms Loss or gain of single entire autosomes are(54.5% vs 45.5%), rate of sperms Loss or gain of part of chromosome are (8.3% vs 0), rate of sperms with Sex chromosome disomy are (16.7% vs 8.3%). None of them show significant difference.Conclusion1. IMSI can improve the rates of normal fertilization in the patients with obstructive azoospermia and teratozoospermia significantly, it can also improve the rate of blastocyst formation rate in the azoospermia patients significantly.2. All the two MSOEM parameters had a significantly correction with the rate of normal morphology sperm and effected on later embryonic development in ICSI cycles; MSOME parameter(the rate of sperm with big head vacuolization which relative area to sperm head>4%), progressive sperm motility and progressive a+b combined motility all had significant effect on thebiochemical pregnancies outcome and MSOME parameters combined the other influencing factors have a predictive value for biochemical pregnancies outcome. In the present study we proposed a cut off value of MSOME parameter(the rate of sperm with big head vacuolization which relative area to sperm head>4%), and demonstrate a relationship between the MSOME parameter and biochemical pregnancies outcome of ICSI cycles. The MSOME parameter could be introduced as an easy diagnostic evaluation prior to perform an ICSI cycle.3.There two groups of sperms sperms with vacuoles(a vacuole area >4%of the head area) and morphological normal sperms show on significant difference at the rate of abnormal molecular karyotypes, however, there is an upward tend of the former. This may because of the limited number of sperms that were analyzed. The MSOME system may reduce the risk of selecting sperm with abnormal karyotypes, it may be the potential mechanism that IMSI can improve the outcomes of patients with poor semen parameters.