of cases 61 45 NA Donor age (Mean ± SD) 26.5 ± 3.0 25.6 ± 2.6 0.45 No. of eggs inseminated 1050 726 NA No. of eggs fertilized (%) 907 (86.4) 645 (88.8) 0.12 No. of blastocysts (%) 587 (64.7) 415 (64.3) 0.88

*Comparison of chromosomal abnormalities in the blastocysts after biopsy by one-step and two-step methods.*

**One-step method Two-step method P value**

**- + P Value**

**Zona hole opening at Day 3 embryo**

observed in the samples with whole chromosome aneuploid rates or segmental abnormalities between two biopsy methods. Samples with multiple chromosomal

*Distribution of chromosomes in the aneuploid blastocysts biopsied with one-step and two-step method. Data represent the number of samples with a single abnormal chromosome and multiple (M:* ≥*2) abnormal* 

abnormalities were also similar between two biopsy methods.

**80**

**Figure 3.**

*chromosomes.*

*NA: Not applicable.*

*NA: Not applicable.*

*embryos at day 3.*

**Table 1.**

**Table 2.**

*Comparison of clinical outcomes after transfer of euploid blastocysts biopsied by one-step and two-step methods from donor egg cycles.*

As shown in **Figure 3**, aneuploidies occurred in all chromosomes except chromosome 12 and the differences for each chromosome were not statistically significant between two methods.

As shown in **Table 3**, transfer of euploid blastocysts biopsied by one-step method had higher chemical pregnancy (78.7 vs. 63.4%), clinical pregnancy (74.5 vs. 61.0%), live birth (70.2 vs. 58.5%) and embryo implantation rates (62.7 vs. 49.2%) as compared with transfer of euploid blastocysts biopsied by two-step method. Although these rates were not statistically significant between two groups, improved clinical outcomes were observed when one-step biopsy method was used.

#### **4. Discussion**

Recently Munne *et al.* found that euploidy rate in human embryos produced by donor egg IVF differed significantly between infertility clinics [12], but they did not analyze the cause(s) related to these differences. Because they collected data from multiple IVF clinics and each clinic used different biopsy methods, which may make it difficult to analyze these factors. In the present study, to minimize the effects of maternal age-related aneuploidy formation in the embryos [6, 13–15, 17–20], we also used donor egg IVF cycles to examine whether biopsy methods affect embryo aneuploidies. Our data indicate that biopsy methods do not affect embryonic aneuploidies, however, simplified biopsy method may improve embryo implantation that may be benefited from reduced embryo manipulations and limited laser applications. We also found that high proportions of human embryos from donor egg IVF are not only whole chromosome aneuploidy, but also have segmental chromosome abnormalities.

Although most of embryonic aneuploidies have already occurred before oocyte and sperm are collected for IVF due to meiotic error(s) during oocyte and sperm development [1–3], some of aneuploidies may be caused by mitotic errors, or suboptimal in vitro conditions and/or in vitro manipulations [1]. Rigorous temperature control during oocyte manipulations can maintain meiotic spindle integrity that may prevent meiosis error during final oocyte maturation after egg retrieval [1]. While embryo biopsy for PGT-A is still an invasive laboratory procedure, thus different methods may affect the embryo quality including chromosome integrity. Since laser was used to zona hole opening and blastocyst biopsy in human IVF, it has made the biopsy procedure to be easy [21]. However, excess use of laser pulses

may be harmful to embryos and eventually would affect embryo development. For example, laser pulse(s) are applied on both cleavage embryos and blastocysts for the traditional two-step biopsy. The blastomeres next to the laser pulses may have different degrees of heat injuring, some injuries can be seen immediately or after further culture, while minor injuries may not be able to see under microscope during culture. Because the biopsied cells are mostly originated from these cells next to the position with laser pulses, chromosomes in some of these cells may be affected, which would eventually increase the rates of chromosomal abnormalities. However, based on our results observed in the present study, these manipulations of embryos do not affect chromosome integrity in the biopsied cells thus the aneuploidies after two-step biopsy were not increased as compared with one-step biopsy method. These results indicate that the chromosome abnormalities during embryo development, if occurs, are not from biopsy procedures.

However, biopsy procedures did affect embryo implantation. Previous studies with non-donor egg IVF found that blastocyst rates and live birth rates were reduced when day 3 zona opening was performed for two-step biopsy [22, 23]. Although we did not find the reduced blastocyst development after day 3 embryo manipulation in the present study, both embryo implantation and live birth rates were reduced when two-step biopsy procedure was used. These results indicate that laser pulses for zona hole opening at day 3 embryos may have detrimental effects on subsequent embryo development and embryo implantation ([22, 23], current study). We did not observe the differences in the blastocyst development after day 3 embryo manipulation in the present study as compared with no day 3 embryo manipulation, which may be due to good quality of oocytes from donors as compared oocytes from patients, thus some blastomeres might be affected, but overall blastocyst development rate was not reduced. Zona hole opening on day 3 embryos by laser pulses may affect embryo development especially if the perivitelline space is small or laser power is too large, thus the detrimental effects were caused by over-heating from laser pulses.

Although the statistical differences of embryo implantation rates between two biopsy methods were not significant due to small cycle numbers in the study by Zhao et al. [22] and in our current study, the differences were significant in the study by Rubino et al. in which more IVF cycles were examined [23]. When we reviewed the clinical outcomes by one-step and two-step biopsy methods in these studies, we found that the overall live birth rates could be increased by approximately 10% (9.26–12.7%) if one-step biopsy method was used, irrespective of small number of cycles or large number of cycles ([22, 23] and the current study) were analyzed.

Another reason for reduced embryo implantation after two-step biopsy may be resulted from blastocyst biopsy procedures. The traditional two-step blastocyst biopsy is performed by mechanical pulling and laser pulses. Heating from laser pulses would also cause injuries to the cells exposed to laser pulses, which would negatively affect embryo quality. Our one-step biopsy procedure is similar as that reported previously [22–24] but some modifications has been made. Cells were aspirated inside zona pellucida that is same as that used by Rubino et al. [23]. However, the cells aspirated into biopsy pipette were separated from blastocyst proper by mechanical blunt dissection, not by mechanical pulling and laser pulses, which is same as that reported by Zhao et al. [24].

The summarized benefits of our method are as the follows: First, one-step method does not need to have embryos to be exposed to laser pulse at day 3, which has been found to be detrimental to blastocyst development [22, 23]. Second, trophectoderm cells are aspirated inside zona pellucida, so that the fertilization of oocytes for PGT-A can be performed by either ICSI or regular IVF, and the contamination by cumulus cells or sperm can be minimized and avoided. Third, the separation of testing cells from blastocyst proper is made by mechanical blunt dissection,

**83**

**5. Conclusions**

*Next-Generation Sequencing Revealed that High Proportion of Human Embryos Resulted…*

not by mechanical suction/pulling and multiple laser pulses, thus the further injuries by laser pulses on isolated cells and blastocyst proper can be avoided. And the last, ICM may hatch from the hole in some blastocysts if zona opening is done at day 3 embryos, thus biopsy need to be done on a different position [23], which

A previous study reported monozygotic twins when two-step biopsy was used [23]. In the present study, we did not observe any monozygotic twins after transfer of blastocysts biopsied by either one-step or two-step method, and this may be attributed to the zona cutting (1/4–1/5) in all frozen blastocysts after warming. We performed blastocyst vitrification after blastocysts were completely collapsed, and the blastocysts were still at collapsed status after warming. The perivitelline space was still very wide after warming, thus laser cutting of a large portion of zona pellucida did not have any injury to the blastocysts. This procedure may avoid

Although biopsy procedures did not affect aneuploid formation in donor egg IVF, the proportions of embryos from donor egg IVF with chromosomal abnormalities are very high [8, 9, 12, 14, 15]. In the present study, we found that these chromosome abnormalities include the whole chromosome aneuploidies and segmental chromosome abnormalities. It has been estimated that ~32% of segmental abnormalities are originated from meiosis [25]. However, most segmental abnormalities originate from mitosis and are present in a mosaic pattern [25, 26]. It has been found that segmental abnormalities can occur in any chromosome, and the

It has been reported that approximately ~6–15% of blastocysts from human IVF have segmental abnormalities when evaluated by current PGT-A methods with different analysis platforms [25, 27, 28]. The incidence of blastocysts with only segmental abnormalities is about 2.4%–7.5% of all samples examined [25, 27–29]. However, in the present study, the segmental chromosome abnormalities accounts for approximately 20–40% of the abnormalities, or around 6–12% of all samples examined, which were higher than previous reports [25, 27, 28]. These differences may be attributed to different PGT analysis platforms because the resolutions and accuracies are different between platforms. The high-resolution PGS platforms, such as NGS, can detect more small chromosome errors than previous microarray and low-resolution platforms. This may also be explanation that PGT by NGS improves pregnancy outcomes compared with array comparative genomic hybrid-

It has been reported that the incidence of segmental abnormalities in human embryos do not correlate with patient age [25, 27, 28]. This may be the reason that high rates were observed in the embryos derived from young and healthy egg donors. Transfer of these embryos would result in failed implantation, miscarriage, or possibly liveborn congenital syndromes if carried to term [29, 30]. Some syndromes and conditions may be related to development delay and intellectual disabilities such as 1q21.1 deletion syndrome, 16p11.2 deletion syndrome and 1p36 deletion syndrome [31], thus screening of these syndromes that have small segmen-

In conclusion, the outcomes from two previous studies [22, 23] and the current study indicate that one-step blastocyst biopsy can improve blastocyst implantation rate and live birth rate by ~10% in non-donor IVF patients [22, 23] and donor egg IVF patients (current study), suggesting that one-step biopsy method is superior

*DOI: http://dx.doi.org/10.5772/intechopen.95457*

monozygotic twins after blastocyst biopsy.

would further affect embryo's implantation competence.

frequency of deletions and duplications is roughly equal [25].

ization in single thawed euploid embryo transfer cycles [16].

tal chromosome abnormalities may be necessary in human IVF.

#### *Next-Generation Sequencing Revealed that High Proportion of Human Embryos Resulted… DOI: http://dx.doi.org/10.5772/intechopen.95457*

not by mechanical suction/pulling and multiple laser pulses, thus the further injuries by laser pulses on isolated cells and blastocyst proper can be avoided. And the last, ICM may hatch from the hole in some blastocysts if zona opening is done at day 3 embryos, thus biopsy need to be done on a different position [23], which would further affect embryo's implantation competence.

A previous study reported monozygotic twins when two-step biopsy was used [23]. In the present study, we did not observe any monozygotic twins after transfer of blastocysts biopsied by either one-step or two-step method, and this may be attributed to the zona cutting (1/4–1/5) in all frozen blastocysts after warming. We performed blastocyst vitrification after blastocysts were completely collapsed, and the blastocysts were still at collapsed status after warming. The perivitelline space was still very wide after warming, thus laser cutting of a large portion of zona pellucida did not have any injury to the blastocysts. This procedure may avoid monozygotic twins after blastocyst biopsy.

Although biopsy procedures did not affect aneuploid formation in donor egg IVF, the proportions of embryos from donor egg IVF with chromosomal abnormalities are very high [8, 9, 12, 14, 15]. In the present study, we found that these chromosome abnormalities include the whole chromosome aneuploidies and segmental chromosome abnormalities. It has been estimated that ~32% of segmental abnormalities are originated from meiosis [25]. However, most segmental abnormalities originate from mitosis and are present in a mosaic pattern [25, 26]. It has been found that segmental abnormalities can occur in any chromosome, and the frequency of deletions and duplications is roughly equal [25].

It has been reported that approximately ~6–15% of blastocysts from human IVF have segmental abnormalities when evaluated by current PGT-A methods with different analysis platforms [25, 27, 28]. The incidence of blastocysts with only segmental abnormalities is about 2.4%–7.5% of all samples examined [25, 27–29]. However, in the present study, the segmental chromosome abnormalities accounts for approximately 20–40% of the abnormalities, or around 6–12% of all samples examined, which were higher than previous reports [25, 27, 28]. These differences may be attributed to different PGT analysis platforms because the resolutions and accuracies are different between platforms. The high-resolution PGS platforms, such as NGS, can detect more small chromosome errors than previous microarray and low-resolution platforms. This may also be explanation that PGT by NGS improves pregnancy outcomes compared with array comparative genomic hybridization in single thawed euploid embryo transfer cycles [16].

It has been reported that the incidence of segmental abnormalities in human embryos do not correlate with patient age [25, 27, 28]. This may be the reason that high rates were observed in the embryos derived from young and healthy egg donors. Transfer of these embryos would result in failed implantation, miscarriage, or possibly liveborn congenital syndromes if carried to term [29, 30]. Some syndromes and conditions may be related to development delay and intellectual disabilities such as 1q21.1 deletion syndrome, 16p11.2 deletion syndrome and 1p36 deletion syndrome [31], thus screening of these syndromes that have small segmental chromosome abnormalities may be necessary in human IVF.

#### **5. Conclusions**

In conclusion, the outcomes from two previous studies [22, 23] and the current study indicate that one-step blastocyst biopsy can improve blastocyst implantation rate and live birth rate by ~10% in non-donor IVF patients [22, 23] and donor egg IVF patients (current study), suggesting that one-step biopsy method is superior

*Cytogenetics - Classical and Molecular Strategies for Analysing Heredity Material*

ment, if occurs, are not from biopsy procedures.

which is same as that reported by Zhao et al. [24].

may be harmful to embryos and eventually would affect embryo development. For example, laser pulse(s) are applied on both cleavage embryos and blastocysts for the traditional two-step biopsy. The blastomeres next to the laser pulses may have different degrees of heat injuring, some injuries can be seen immediately or after further culture, while minor injuries may not be able to see under microscope during culture. Because the biopsied cells are mostly originated from these cells next to the position with laser pulses, chromosomes in some of these cells may be affected, which would eventually increase the rates of chromosomal abnormalities. However, based on our results observed in the present study, these manipulations of embryos do not affect chromosome integrity in the biopsied cells thus the aneuploidies after two-step biopsy were not increased as compared with one-step biopsy method. These results indicate that the chromosome abnormalities during embryo develop-

However, biopsy procedures did affect embryo implantation. Previous studies with non-donor egg IVF found that blastocyst rates and live birth rates were reduced when day 3 zona opening was performed for two-step biopsy [22, 23]. Although we did not find the reduced blastocyst development after day 3 embryo manipulation in the present study, both embryo implantation and live birth rates were reduced when two-step biopsy procedure was used. These results indicate that laser pulses for zona hole opening at day 3 embryos may have detrimental effects on subsequent embryo development and embryo implantation ([22, 23], current study). We did not observe the differences in the blastocyst development after day 3 embryo manipulation in the present study as compared with no day 3 embryo manipulation, which may be due to good quality of oocytes from donors as compared oocytes from patients, thus some blastomeres might be affected, but overall blastocyst development rate was not reduced. Zona hole opening on day 3 embryos by laser pulses may affect embryo development especially if the perivitelline space is small or laser power is too large,

thus the detrimental effects were caused by over-heating from laser pulses.

Although the statistical differences of embryo implantation rates between two biopsy methods were not significant due to small cycle numbers in the study by Zhao et al. [22] and in our current study, the differences were significant in the study by Rubino et al. in which more IVF cycles were examined [23]. When we reviewed the clinical outcomes by one-step and two-step biopsy methods in these studies, we found that the overall live birth rates could be increased by approximately 10% (9.26–12.7%) if one-step biopsy method was used, irrespective of small number of cycles or large number of cycles ([22, 23] and the current study) were analyzed. Another reason for reduced embryo implantation after two-step biopsy may be resulted from blastocyst biopsy procedures. The traditional two-step blastocyst biopsy is performed by mechanical pulling and laser pulses. Heating from laser pulses would also cause injuries to the cells exposed to laser pulses, which would negatively affect embryo quality. Our one-step biopsy procedure is similar as that reported previously [22–24] but some modifications has been made. Cells were aspirated inside zona pellucida that is same as that used by Rubino et al. [23]. However, the cells aspirated into biopsy pipette were separated from blastocyst proper by mechanical blunt dissection, not by mechanical pulling and laser pulses,

The summarized benefits of our method are as the follows: First, one-step method does not need to have embryos to be exposed to laser pulse at day 3, which has been found to be detrimental to blastocyst development [22, 23]. Second, trophectoderm cells are aspirated inside zona pellucida, so that the fertilization of oocytes for PGT-A can be performed by either ICSI or regular IVF, and the contamination by cumulus cells or sperm can be minimized and avoided. Third, the separation of testing cells from blastocyst proper is made by mechanical blunt dissection,

**82**

to two-step method. Although blastocyst biopsy procedures may not affect the incidence of aneuploidies and/or segmental chromosome abnormalities, they affect embryo's implantation competences. Current PGT by high-resolution NGS reveals that high proportions of human embryos derived from donor eggs are not only whole chromosome aneuploidies, buy also segmental abnormal. Therefore, screening of these chromosome abnormalities may reduce embryo implantation failure, early miscarriage, birth defect, developmental delay and/or intellectual disability.
