Effects Of Two-step First And Second Polar Body Biopsy On Fertilization Of Oocyte And Embryo Viability In A Mouse Model

Preimplantation genetic diagnosis (PGD) was introduced as an option for avoiding selective abortion following prenatal diagnosis by chorionic villus sampling (CVS) or amniocentesis. PGD has been offered to hundreds of couples at high risk for having children with genetic and chromosomal disorders. In most cases, PGD has been preformed by blastomere biopsy and resulted in the birth of many children free of genetic diseases. But incidence of chromosome mosaicism in cleavage-stage embryos was high, which resulted in misdiagnosis. Blastomere biopsy was high invasive. To avoid embryo biopsy at the cleavage stage which may affect the viability of the embryo, PGD by the first polar body (lPB) analysis has been introduced and was shown to be the method of choice in predicting the genotype of the oocytes. However, the genotype of a considerable number of embryos resulting from oocytes diagnosed as heterozygous by the lPB analysis was not predictable without of test of their second PB (2PB). Due to recombination at meiosis as much as half of 1PBs might be heterozygous. To ensure that hemizygous normal oocytes following the second meiotic division can be detected and replaced, we introduced the 2PB analysis as an integral part ofpreimplantation polar body diagnosis. PBs were not necessary to embryo development and zygote formation, so PB biopsy could not result in reduction of embryonic component and not affect embryo viability. Security of biopsy in PGD is still an issue that most people paid attention to. The security and reliability of blastomere biopsy has been approved. But whether or not, has PB biopsy, especially two-step IPB and 2PB biopsy, adverse effect on fertilization and embryo viability, and was it more secure compared with blastomere biopsy? There is no report in our country. The recent development of a non-contact 1.48 μ mdiode laser system offers an excellent method for zona drilling in biopsy. Since laser drilling started to be used in PGD from 1997, this laser system has been a more convenient, secure, precise biopsy-assistant tool. In our research with mice, the effects on embryo viability between different biopsy protocols were compared. The purpose of this study was to evaluate security of two-step PB biopsy, and to try to provide the basic rationale and the best protocol for clinical applications.Materials40 Kunming white male mice at the age of 8-12 weeks and 60 female mice at the age of 8-10 weeks were provided from Henan Laboratory Animal Center. Bright/dark cycle in the environment the mice living in was 10h/14h. The mice drank and ate without time limit.Methods1. Females were superovulated by i.p. injection of 10 IU HMG followed 48h later by 10 IU of hCG. Females were killed at 14 h after hCG injection. Oviducts were excised and placed into 2 ml of HEPES medium supplemented with 10% SPS. Ovulated cumulus-enclosed and cumulus-free oocytes were released from the ampullae. Mucous cumulus cells were removed by gentle agitation with 80 IU/ml hyaluronidase. After removing cumulus cells, oocyteswere washed several times and left for 30min in fertilization medium supplemented with 10% SPS overlaid with mineral oil at 37°C in a humidified atmosphere of 6% CO2 in air. Oocyte insemination was performed with ICSI. Only the oocytes with integral 1PB were included in this study. At 4-5h after ICSI, fertilization was evaluated. Zygote cleavage were observed at 24h and 48h after fertilization. Resulting embryos were grading. Within 96h to 144h after fertilization, blastocyst formation and hatching were evaluated.2. All the oocytes were divided into eight groups according to different micromanipulation. Group 1, laser drilling after ICSI; Group 2, laser drilling and lPB biopsy after ICSI; Group 3, laser drilling and lPB biopsy after ICSI, and 2PB biopsy after fertilization from the solo zona opening; Group 4, laser drilling after fertilization; Group 5, laser drilling and simultaneous lPB and 2PB biopsy after fertilization; Group 6, laser drilling at 6- to 8-cell stage; Group 7, laser drilling and one blastomere biopsy at 6- to 8-cell stage; Group 8, control group, ICSI only.3. Compare fertilized oocyte, cleaved zygote, good embryo, resulting blastocyst, blastocyst quality, hatching blastocyst and hatched blastocyst rate between groups.Results1. Effects of laser drilling at different stage on fertilization of mouse oocyte and embryo viabilityIn each group of laser drilling, fertilized oocyte rate, cleaved zygote rate, 4-cell rate, 8-cell rate and good embryo rate were not significantly different from those in the control. Grade Ⅰ , grade Ⅱ , grade Ⅲ blastocyst rate, total resulting blastocyst rate and expanding blastocyst rate were similar to those in the control. The proportion of hatching blastocyst in the group of drilling after ICSI (88.6%) was significantly higher than that in the control (64.7%), P<0.05. This proportionin the group of drilling after fertilization (90.0%) or at 8-cell stage (91.1%) was also significantly higher than that in the control (64.7%), P<0.01. The proportion of complete hatching blastocyst out of expanding blastocyst in each group of laser drilling was 62.9%, 62.5%, 62.2%, respectively. Each was significantly higher than that in the control (41.2%), P<0.05. But the proportion of complete hatching blastocyst out of initial hatching blastocyst (71.0%, 71.4%, 68.3%) was not statistically different from that in the control (63.6%). In the three drilling groups, each rate above was similar to each other.2. Effects of lPB biopsy after ICSI or and 2PB biopsy after fertilization on fertilization of mouse oocyte and embryo viability2.1 Only lPB biopsy after ICSI on fertilization of oocyte and embryo viability Fertilized oocyte rate and cleaved zygote rate in the group of lPB biopsy afterICSI were 69.1% and 81% respectively, which were lower than those in the control (76.4% and 88.7%), but there was no statistic significance between the two groups. 4-cell and 8-cell rate, high grade embryo rate were not significantly different from those in the control. Grade I, grade II, grade III blastocyst rate were similar to those in the control. Total resulting blastocyst rate (52.9%) was lower than that in the control (60.4%), but there was no statistic significance between the two groups. Expanding blastocyst rate (83.8%) was not significantly different from that in the control (79.7%). Hatching blastocyst rate (87.1%) was significantly higher than that in the control (64.7%), f<0.05. The proportion of complete hatching blastocyst out of expanding blastocyst (58.1%) was higher than that in the control (41.2%), but there was no statistic significance. The proportion of complete hatching blastocyst out of initial hatching blastocyst (66.7%) was similar to that in the control (63.6%). Compared with the group of drilling after ICSI, each rate above was similar.2.2 Two-step lPB and 2PB biopsy after ICSI and fertilization respective onfertilization of oocyte and embryo viabilityIn the group of 1PB biopsy after ICSI and 2PB biopsy after fertilization, 184 matured MII oocytes were removed lPB after ICSI, 129 oocytes were fertilized, the fertilization rate was 70.1%. 108 out of 129 had cleaved into 2-cell embryos, and 2PB was observed clearly. But only 101 out of 108 were successfully biopsied. The efficiency was 93.5%. Fertilized oocyte, cleaved zygote, 4-cell, 8-cell and high grade embryo rate were a little lower than those in the control. But there was no statistic significance between the two groups. Grade I blastocyst rate (8.9%) and total resulting blastocyst rate (50.7%) were lower than those in the control (12.3%, 60.4%), but there was no statistic significance between the two groups. Expanding blastocyst rate was similar to that in the control. Hatching blastocyst rate (88.9%) was significantly higher than that in the control (64.7%), P<0.05. The proportion of complete hatching blastocyst out of expanding blastocyst (55.6%) was higher than that in the control (41.2%), but there was no statistic significance. The proportion of complete hatching blastocyst out of initial hatching blastocyst (62.5%) was similar to that in the control (63.6%). Compared with the group of drilling after ICSI, Grade II blastocyst rate and total resulting blastocyst rate were a little lower, but there was no statistic significance between the two groups. Compared with the group of only lPB biopsy, each rate above was similar. 3. Effects of simultaneous lPB and 2PB biopsy after fertilization on fertilization of mouse oocyte and embryo viabilityOf the 128 fertilized oocytes in the group of simultaneous lPB and 2PB biopsy after fertilization, 115 fertilized oocytes had cleaved into 2-cell embryos, and lPB and 2PB were observed clearly. Only 106 out of 115 were successfully biopsied. The efficiency was 92.2%. Compared with those in the control or in the group of drilling after fertilization, 4-cell, 8-cell, high grade embryo rate and grade I, grade II blastocyst rate were a little lower, but there was no statistic significancebetween groups. Total resulting blastocyst rate (52.2%) was also lower than that in the control (60.4%) and that in the group of drilling (61.3%), but there was no statistic significance between groups. Compared with that in the control, expanding blastocyst rate was similar. Hatching blastocyst rate was significantly higher (93.1%, 64.7%, P<0.01). The proportion of complete hatching blastocyst out of expanding blastocyst was also higher, but there was no statistic significance (58.6%, 41.2%, p>0.05). The proportion of complete hatching blastocyst out of initial hatching blastocyst was similar.4. Effects of blastomere biopsy at 6- to 8-cell stage on fertilization of mouse oocyte and embryo viabilityFertilized oocyte, cleaved zygote, 4-cell, 8-cell and high grade embryo rate were not significantly different from those in the control. Of 82 high grade embryos, only 75 embryos were successfully removed one blastomere, biopsy efficiency was 91.5%. Grade I blastocyst rate (6.7%) and total resulting blastocyst rate (46.7%) were lower than those in the control (12.3%, 60.4%), but there was no statistic significance between the two groups. Compared with that in the control, expanding blastocyst rate was similar. Hatching blastocyst rate was significantly higher (92.6%, 64.7%, P<0.01). The proportion of complete hatching blastocyst out of expanding blastocyst was also higher, but there was no statistic significance (51.8%, 41.2%, P>0.05). The proportion of complete hatching blastocyst out of initial hatching blastocyst was similar. Each rate above was similar to that in the group of drilling at 6- to 8-cell stage.5. Effects of sequential 1PB and 2PB biopsy on fertilization of mouse oocyte and embryo viability compared with that of blastomere biopsyFertilized oocyte rate and cleaved zygote rate, 4-cell and 8-cell rate, high grade embryo rate and resulting blastocyst rate between the three groups were similar to each other. Expanding blastocyst rate, hatching blastocyst rate, hatched blastocystrate were also not significantly different from each other. Conclusion1. Zona drilling by laser had no adverse impact on fertilization of oocytes and the viability of resulting embryos. It also had no adverse effect on blastocyst formation, whereas it might be helpful to improve blastocyst hatching. And the effects of laser drilling at different development stage were similar. This laser shot system is a precise, convenient, secure tool for zona drilling in biopsy of polar body or blastomere.2. As the time of appearance of lPB and 2PB were different, two kinds of protocols of sequential lPB and 2PB biopsy were devised, one was two-step lPB and 2PB biopsy after maturation and fertilization respective, the other was simultaneous lPB and 2PB biopsy after fertilization. Besides, there was a little distance between lPB and 2PB, so it had a little difficulty in lPB and 2PB biopsy from solo zona opening by laser drilling. But our study proves that the two protocols are feasible.3. Both the two protocols of sequential lPB and 2PB biopsy had no adverse impact on fertilization of mouse oocytes and the viability of resulting embryos, whereas they might be helpful to improve blastocyst hatching. Two-step lPB and 2PB biopsy is a secure biopsy procedure in PGD. Moreover, two-step PBs biopsy has more advantage and higher accuracy of diagnosis, thus it should be believed as the best protocol of PBs biopsy.