The Study On The Value Of 0PN/1PN-Derived Embryos In Conventional IVF Cycles

In in vitro fertilization(IVF) or intracytoplasmic sperm injection(ICSI) treatment cycles, a fertilization check is performed at 16-20 hours post insemination to exclude oocytes that have fertilized abnormally or not fertilized at all. Normal fertilization of an oocyte is defined by observing two distinct pronuclei(2PN) and two polar bodies after insemination. Oocytes showing no pronucleus(0PN), one pronucleus(1PN) or three pronuclei(3PN)(or more) are deemed as having fertilized abnormally and may be discarded. However, this assumption may not always be accurate. Cytogenetic analysis has revealed that a proportion of diploid embryos can develop from 1PN or 0PN zygotes. Healthy babies developing from such embryos have been reported. Furthermore, there are reports of 0PN-derived embryos present in 12% to 20% of Day 3 embryos. In the IVF cycle, The incidence of 1PN was 2.7-5.5% in IVF cycle, and 4.9-11.4% in ICSI cycle. 1PN or 0PN derived embryos may be the only hope of obtaining a pregnancy for the patients with only few oocytes, such as the elder or the patients with ovarian dysfunction. Therefore, it is necessary for us to make a study on the possible factors affecting the occurrence of 0PN/1PN-derived embryos, chromosome analysis, embryo quality and clinical outcomes of 1PN and 0PN derived embryos.To make more objective judgment on the chromosome status of 0PN and 1PN derived embryos, genetic screening preimplantation(PGS) is needed to be performed in embryos. Two kinds of gene chip, namely microarray comparative genomic hybridization analysis(a CGH) and single nucleotide polymorphism microarray technology(SNP array) are widely used to detect chromosomal abnormalities. But the gene chip can only detect the limited variation within the known sequences. Meanwhile, because of its high cost, complex operation technology, to a certain extent, chip technology has been limited to further popularity. The next generation sequencing technology(NGS) is a comprehensive reading of the whole genome of nucleic acids, therefore, it is an open system, and can be repeated several times. Therefore, in essence, the ability to discover new information and the ability to find new information is higher than the gene chip technology. Many studies have confirmed that NGS is effective in the diagnosis of chromosomal abnormalities in oocytes and embryos. There is no report on detection of chromosome ploidy by NGS technology.Therefore, binary logistic analysis were performed looking at the relationship of the occurrence of 0PN/1PN-derived embryos and the covariates. To detect the value of 0PN/1PN embryos by comparing the outcomes of transferring embryos derived from different number of pronucleus. Through the comparison the outcomes of cleavage embryos and blastcysts to detect the selective role of blastcyst culture on 0PN/1PN embryos. To investigate the predictive effect of 1PN pronucleus diameter on the quality of embryo by comparing cleavage stage embryo quality and blastocyst formation rate of 1PN zygotes with different pronucleus diameter. By the next generation sequencing technology to detect the aneuploidy rate of 0PN/1PN-derived blastocyst, compared with that of 2PN blastocyst, for providing theoretical support for the clinical application value of 1PN, 0PN derived blastocyst.Part I The study on the value of 0PN-derived embryos in conventional IVF cycles Objective:The aim of this study was to 1) investigate the incidence and related factors of embryos derived from “unfertilized oocytes” i.e.oocytes not displaying pronuclei(0PN) at the time of the fertilization check, and 2) the clinical pregnancy rates when transferring 0PN-derived embryos. Methods:1. A retrospective analysis of clinical treatment outcome of fresh cycle with cleavage stage embryo transfer:From Jan 2007 to Jun 2014, all embryo transfers from conventional IVF treatment were included in this study. A binary logistic analysis was performed looking at the relationship of the occurrence of 0PN-derived embryos and the covariates. Binary logistic analysis was performed to analyze the impact of cycle characters and the transferring of embryos derived from different number of pronucleus on clinical pregnancy rate and live-birth rates. According to different sources of embryos, all embryo transfer(ET) cycles were divided into 0PN ET group(70 cycles), 0PN+2PN ET group(113 cycles) and 2PN ET group(2628 cycles). To compare female’s age, number of oocytes per cycle, number of embryo transferred, clinical pregnancy rate, implantation rate of all the groups.2. A retrospective analysis of blastocyst transfer in FET(frozen-thawed ET) cycles:From January 2014 to June 2015, all blastocyst derived from conventional IVF treatment in FET cycles were included in this study. According to the source of the blastocyst transferred, all cycles were divided into cycles with 2PN derived blastocyst transferred and cycles with 0PN derived blastocyst transferred. The clinical pregnancy rate, implantation rate and abortion rate of the two groups were compared. Results:1. In the study period from 2007 to 2014, 4424 IVF-ET cycles were reviewed. It was noted that 11.3%(4966/43949) of embryos observed were scored as 0PN. Two hundred and seventy five embryos that developed from 0PN oocytes with embryo grade III or better were transferred in 183 cycles. Of these 183 treatment cycles, 70 cycles had embryos transferred where 0PN-derived embryos only were available. The CPR of the cycles where 0PN-derived embryos were exclusively transferred was 24.3%(17/70). The implantation rate was 17.0%(19/112) with 1 ectopic pregnancy, 2 miscarriages and 1 stillbirth observed. Of the 13 healthy infants born from 13 cycles, there were no reports of deformities.2. Clinical features of cycles with and without 0PN-derived embryo are compared. Cycles where 0PN-derived embryos did occur had significantly lower female age, FSH level, and higher number of oocytes retrieved. Although a significantly lower 2PN rate was observed in the cycles where 0PN-derived embryos were present, the top-quality embryo rate was significantly higher than those cycles without 0PN-derived embryos. It was found that female age, number of oocytes and the top-quality embryo rate were significantly correlated with the the occurrence of 0PN-derived embryo(B=1.01, Wald=16.29, P=0.00). Adjusted by female age, BMI, the top-quality embryo rate and number of embryos transferred, the source of embryos transferred, i.e. 0PN-derived embryos or 2PN embryos, did not have a significant impact on clinical pregnancy rate(CPR)and live-birth rate(LBR).3. The age of patients of 0PN ET group(33.05±5.84)was older than 2PN ET group(31.36±5.09), the difference was significant, P < 0.05; there was no significant difference between 0PN ET group and 0PN+2PN ET group in female’s age(31.36±5.09), P > 0.05. The average number of oocytes per cycle(6.49±4.43 VS 9.57±4.94), average number of transferred embryos(1.62±0.58 VS 2.03±0.44), clinical pregnancy rate(24.3% VS 46.4%)and implantation rate(17.0% VS 29.4%)of 0PN ET group were significantly lower than those of 2PN ET group, the difference was significant, P < 0.05. The average number of oocytes per cycle, the average number of transferred embryos and clinical pregnancy rate in 0PN ET group were significantly lower than those in 0PN+2PN ET group(9.28±4.44,2.21±0.41,40.7%,22.3%), the difference was significant, P < 0.05. The implantation rate of 0PN+2PN ET group was lower than that of 2PN group ET(22.3% VS 29.4%), the difference was significant, P < 0.05.4. There was no significant difference in the age, the thickness of endometrium and the average number of embryos transferred between the 0PN blastocyst FET group and the 2PN blastocyst FET group, P > 0.05, which indicated that the two groups were comparable. No significent difference were found in clinical pregnancy rate and implantation rate between the two groups, P > 0.05, but the abortion rate of 0PN blastocyst FET group was higher than the 2PN blastocyst FET group(17.6% VS 5.8%), the difference was significant, P < 0.05. Conclusions:1. The appearance of 0PN-derived embryos in IVF can be considered as an indicator of a cycle with appropriate embryo development.2. IVF derived 0PN embryos can achieve similar clinical pregnancy rate and live birth rate with 2PN.3. Blastcyst culture can improve the clinical pregnancy rate per cycle. Part II The study on the value of 1PN-derived embryos in conventional IVF cycles Objective:The aim of this study was to 1) investigate the incidence and related factors of embryos derived from monopronucleated(1PN) zygotes at the time of the fertilization check, and 2) investigate the predictive effect of 1PN pronucleus diameter on the quality of embryo, and 3)the clinical outcome when transferring 1PN-derived embryos. Methods:1. A retrospective analysis of clinical treatment outcome of fresh cycle with cleavage stage embryo transfer:From Jan 2007 to Jun 2015, all embryo transfers from conventional IVF treatment were included in this study. Women of all ages were included. The karyotypes of the couple were normal. A binary logistic analysis was performed looking at the relationship of the occurrence of 1PN-derived embryos and the covariates. According to the pronucleus diameter, 1PN zygotes were divided into 3 groups. To compare cleavage stage embryo quality and blastocyst formation rate of all the groups. According to different sources of embryos, all embryo transfer(ET) cycles were divided into 1PN ET group(20 cycles), 1PN+2PN ET group(51 cycles) and 2PN ET group(3750 cycles). To compare female’s age, number of oocytes per cycle, number of embryo transferred, clinical pregnancy rate, implantation rate of all the groups.2. A retrospective analysis of blastocyst transfer in FET(frozen-thawed ET)cycles:From January 2014 to June 2015, all blastocyst derived from conventional IVF treatment in FET cycles were included in this study. According to the source of the blastocyst transferred, the cycles were divided into cycles with 2PN derived blastocyst transferred(1040cycles) and cycles with 1PN derived blastocyst transferred(22cycles). The clinical pregnancy rate, implantation rate and abortion rate of the two groups were analyzed. Results:1. In the study period from 2007 to 2015, 6434 IVF-ET cycles were reviewed. It was noted that 7.5%(3916/51922) of embryos observed were scored as 1PN.2. the study on the occurrence of 0PN-derived embryosClinical features of cycles with and without the incidence of 1PN-derived embryos are compared. Cycles where 1PN-derived embryos did occur had significantly lower female age(30.79±5.25 VS 33.03±6.22), FSH level(6.71±2.38 VS 7.74±3.94), higher number of eggs retrieved(13.82±7.19 VS 8.32±5.88), and lower 2PN rate(61.1% VS 66.6%). But rate of top-quality embryos, clinical pregnancy rate, miscarriage rate and live-birth rate were found no statistically difference(P>0.05). It was found that basal FSH, number of oocytes and 2PN rate were significantly correlated with 1PN-derived embryo.3. Comparison of ET results from different type of cleavage embryosThere was no significant difference between 1PN ET group and 2PN ET group in female’s age and average number of oocytes per cycle. Average number of transferred embryos(1.20±0.42 VS 1.97±0.44), clinical pregnancy rate(10.0% VS 51.7%)and implantation rate(8.3% VS 36.8%)of 1PN ET group were significantly lower than those of 2PN ET group, the difference was significant, P < 0.05. There was no significant difference between 1PN ET group and 0PN+2PN ET group in female’s age, average number of oocytes per cycle, average number of transferred embryos, clinical pregnancy rate and implantation rate. There were significant difference between 1PN+2PN ET group and 2PN ET group in female’s age( 33.73±4.73 VS 31.23±7.22), average number of oocytes per cycle( 8.53±5.12 VS 10.03±5.13), average number of transferred embryos( 2.37±0.49 VS 1.97±0.44), clinical pregnancy rate( 31.4% VS 51.7%), and implantation rate( 21.5% VS 36.8%).4. Comparison of FET results from different type of blastcystsThere was no significant difference in the age and endometrial thickness between the 1PN blastcyst group and 2PN blastcyst group in FET cycle. The average number of embryos transferred of 1PN blastcyst group was significantly less than that of 2PN blastcyst group(1.09±0.23 VS 1.74±0.38), the difference was significant, P < 0.05. The clinical pregnancy rate(27.3% VS 76.2%) and implantation rate(25.0% VS 54.4%) of 1PN blastcyst group were lower than that of 2PN blastcyst group, the difference was significant, P < 0.05. Six of 22 cycles were clinical pregnancy with no abortion in 1PN blastcyst group.5. The relationship of pronucleus diameter and embryo qualityAccording to the pronucleus diameter, 1PN zygotes were divided into 3 groups, that is, ≥29μm group,26-28 μm group and ≤25μm group. The 6-8 cell rate(95.7% VS 66.7%), rate of embryo with fragmentation <20%(82.6% VS 38.5%) and blastocyst formation rate(65.2% VS 27.8%) of ≥29μm group was higher than that of 26-28 μm group and ≤25μm group(44.7%,45.8%,16.7%), the difference was significant, P < 0.05. The 6-8 cell rate and blastocyst formation rate of 26-28 um group was higher than that of ≤25μm group, the difference was significant, P < 0.05. The total blastocyst formation rate of 1PN embryos was 25.3%(133/526). Conclusions:IVF 1PN cleavage stage embryos and blastocyst because of its poor development potential.Part III The study of aneuploidy of 0PN/1PN-derived blastocyst using next generation sequencing Objective:To compare the blastocyst formation rate among 0PN, 1PN and 2PN derived embryo. By the next generation sequencing technology to detect the aneuploidy of 0PN/1PN-derived blastocyst, compared with that of 2PN blastocyst from the genetic point of view, for providing theoretical support for the clinical application value of 1PN, 0PN derived blastocyst. Methods:From January 2015 to August, all embryo transfers from conventional IVF treatment were included in this study. Inclusion criteria: the female’s age ≤ 35 years, both the male and the female have normal chromosome. All 0PN and 1PN derived embryos were collected during the period, and were cultured to the blastocyst stage. 2PN derived embryos in the same period were defined as control group. To compare the blastocyst formation rate of embryos with different number of pronucleus nuclei. Twenty 2PN blastocysts performed with PGD preliminary experiment were collected and defined as control group in reproductive medical center of the third affiliated hospital of zhengzhou university. Twenty 0PN blastocysts and twenty 1PN blastocysts were selected for blastocyst biopsy. Samples were performed with whole chromosoms screening by next generation sequencing and compare the aneulpoidy rates of the 3 groups. Results:The blastocyst formation rates of 2PN, 1PN and 0PN embryo were 52%, 25.3% and 47.9%, respectively. The blastocyst formation rate of 2PN was higher than 0PN group and 1PN group, and the difference was significant, P < 0.05. Compared with the blastocyst formation rate between 1PN and 0PN embryo, the difference was significant, P < 0.05. The chromosome aneuploidy of 2PN, 1PN and 0PN derived blastocyst were 40%, 70% and 40%, respectively. There was no significent difference between 0PN and 2PN embryo, but significent difference was found between 1PN and 2PN embryo. Conclusions:1. IVF derived 0PN blastocyst had similar chromosomes aneulpoidy rates with 2PN blastocyst.2. IVF derived 1PN blastocyst had high chromosomes aneulpoidy rates.