**2. Materials and methods**

#### **2.1 Ethical statement**

The patients signed the consents for all laboratory and clinical procedures including embryo biopsy for PGT-A. The data was collected from medical records at the clinic and laboratory, and the study with PGT-A was approved by New England Institutional Review Board (NEIRB 14–504).

#### **2.2 Donor stimulation**

Donors for IVF treatment underwent controlled ovarian stimulation with a combination of daily injection of 75–300 IU recombinant follicle-stimulating hormone (Gonal-F, EMD Serono, MA, USA) and 75–300 IU of a combination of follicle stimulating hormone and luteinizing hormone (Menopur, Ferring Pharmaceuticals, NJ, USA). On day 5–7, 0.25 mg gonadotropin releasing hormone antagonist (Cetrotide, EMD Serono) was given daily until triggering for oocyte maturation by gonadotropin-releasing hormone agonist (Lupron) or human chorionic gonadotropin (hCG). Oocytes were retrieved at 35–36 hours after the trigger and then cultured in Global™Total medium (Origio Inc., CT, USA) at 37°C in an atmosphere of 5.5% CO2, 6% O2, and balanced N2 under humidified or dry conditions.

#### **2.3 Oocyte insemination and embryo culture**

Oocytes were inseminated by intracytoplasmic sperm injection (ICSI) after cumulus cells were removed by using hyaluronidase (Fujifilm-Irvine Scientific) at 3–4 hours after oocyte retrieval and metaphase II oocytes were injected 4–5 hours after retrieval. After insemination, oocytes were cultured in Global™Total medium at 37°C in an atmosphere of 5.5% CO2, 6% O2, and balanced N2 under humidified or dry conditions.

Fertilization was assessed 16–18 hours after insemination, and normal fertilization was characterized by two distinct pronuclei and two polar bodies. Fertilized

**77**

**Figure 1.**

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

oocytes were further cultured in the Global™Total medium and embryo quality was

Two biopsy methods were used in the present study. The first is a traditional two-step method in which a small hole in zona pellucida was opened by laser pulses on cleavage embryos at Day 3. As shown in **Figure 1**, when embryo developed to blastocyst at day 5 or later and some cells from blastocysts hatched from the hole, 5–10 cells were aspirated to a biopsy pipette and then cells were separated from

The second is a modified and simplified one-step method with less embryo manipulation and less laser application. Hole opening in the zona pellucida was not performed on day 3 embryos. Blastocysts were directly processed for biopsy. The details for this method are as the follows: As shown in **Figure 2**, blastocyst for biopsy was held to a proper position (**Figure 2A**) in which inner cell mass (ICM) was on the 6–9 O'clock position, a small hole in the zona pellucida was opened (**Figure 2B**) on the 3 O'clock position by one laser pulse with the ZILOS-tk™ laser system (Hamilton Thorn Bioscience Inc., MA, USA). A 20 μm polished biopsy pipette (Sunlight Medical, Jacksonville, FL, USA) was inserted to inside zona pellucida through the hole and a few trophectoderm cells on the 12–2 O'clock position were aspired into biopsy pipette (**Figure 2B**). After biopsy pipette was pulled out of the zona (**Figure 2C**), biopsy pipette together with blastocyst was moved to the top the holding pipette (**Figure 2D**), and the biopsy pipette was pull down against the holding pipette so that the cells inside the biopsy pipette were completely separated

After biopsy, biopsied cells were washed individually, transferred to PCR tubes,

and stored at −20°C freezer until processing for PGT-A by commercial genetic testing company. Blastocysts were individually cryopreserved by vitrification for later frozen embryo transfer (FET). Blastocysts were classified as abnormal if they

*Procedures for two-step blastocyst biopsy. A blastocyst with some trophectoderm cells being hatched from the hole in the zona pellucida opened on day 3 and the blastocyst is held to a proper position for biopsy (A). After a few trophectoderm cells are aspirated into biopsy pipette, one laser pulse is applied on upside of the cells (B) and another lase pulse is applied to the bottom side of cells (C) during mechanical pulling. Extra laser pulses may be necessary during pulling until the cells are completely isolated from blastocyst proper (D).*

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

from blastocyst proper (**Figure 2E**).

**2.4 Blastocyst biopsy**

evaluated by an inverted microscope on day 3, 5, or 6.

blastocyst proper by mechanical pulling and laser pulses.

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

oocytes were further cultured in the Global™Total medium and embryo quality was evaluated by an inverted microscope on day 3, 5, or 6.

#### **2.4 Blastocyst biopsy**

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

however, for PGT-A, usually 10 Mb and above are detected and reported.

**2. Materials and methods**

Institutional Review Board (NEIRB 14–504).

**2.3 Oocyte insemination and embryo culture**

**2.1 Ethical statement**

**2.2 Donor stimulation**

aneuploidies (PGT-A) [13–17]. With PGT-A by NGS, not only a whole chromosome aneuploidy can be detected, but also segmental chromosome abnormalities (deletion and duplication) can be detected [18–21]. Segmental chromosome abnormalities typically represent regional losses or gains in one or more chromosomes. The size of a segmental abnormalities detectable by current NGS platforms is as small as 1 Mb,

Some segmental chromosome abnormalities may cause miscarriage and birth defect, while others may result in developmental delay and/or intellectual disability if the transfer of such embryos produce live birth. It has been found that the prevalence of embryonic aneuploidy in donor egg IVF was significantly different between fertility clinics indicating that clinical and laboratory procedures may be related to the occurrence of embryonic aneuploidies [12]. Embryo biopsy is a complicated and invasive laboratory procedure that involves several embryo manipulations during culture, so it may affect embryo's quality including aneuploidies. Therefore, in the present study, to examine whether embryo biopsy procedures affect embryonic aneuploidies in donor egg IVF, blastocysts were biopsied by two different biopsy methods and then the samples were examined by NGS. Collected data were analyzed in terms of the rates of embryos with whole chromosome aneuploidies and segmental chromosome abnormalities. Clinical outcomes, such as pregnant rate, live birth rate and embryo implantation rate were also analyzed.

The patients signed the consents for all laboratory and clinical procedures including embryo biopsy for PGT-A. The data was collected from medical records at the clinic and laboratory, and the study with PGT-A was approved by New England

Donors for IVF treatment underwent controlled ovarian stimulation with a combination of daily injection of 75–300 IU recombinant follicle-stimulating hormone (Gonal-F, EMD Serono, MA, USA) and 75–300 IU of a combination of follicle stimulating hormone and luteinizing hormone (Menopur, Ferring Pharmaceuticals,

(Cetrotide, EMD Serono) was given daily until triggering for oocyte maturation by gonadotropin-releasing hormone agonist (Lupron) or human chorionic gonadotropin (hCG). Oocytes were retrieved at 35–36 hours after the trigger and then cultured in Global™Total medium (Origio Inc., CT, USA) at 37°C in an atmosphere

Oocytes were inseminated by intracytoplasmic sperm injection (ICSI) after cumulus cells were removed by using hyaluronidase (Fujifilm-Irvine Scientific) at 3–4 hours after oocyte retrieval and metaphase II oocytes were injected 4–5 hours after retrieval. After insemination, oocytes were cultured in Global™Total medium at 37°C in an atmosphere of 5.5% CO2, 6% O2, and balanced N2 under humidified or dry conditions. Fertilization was assessed 16–18 hours after insemination, and normal fertilization was characterized by two distinct pronuclei and two polar bodies. Fertilized

NJ, USA). On day 5–7, 0.25 mg gonadotropin releasing hormone antagonist

of 5.5% CO2, 6% O2, and balanced N2 under humidified or dry conditions.

**76**

Two biopsy methods were used in the present study. The first is a traditional two-step method in which a small hole in zona pellucida was opened by laser pulses on cleavage embryos at Day 3. As shown in **Figure 1**, when embryo developed to blastocyst at day 5 or later and some cells from blastocysts hatched from the hole, 5–10 cells were aspirated to a biopsy pipette and then cells were separated from blastocyst proper by mechanical pulling and laser pulses.

The second is a modified and simplified one-step method with less embryo manipulation and less laser application. Hole opening in the zona pellucida was not performed on day 3 embryos. Blastocysts were directly processed for biopsy. The details for this method are as the follows: As shown in **Figure 2**, blastocyst for biopsy was held to a proper position (**Figure 2A**) in which inner cell mass (ICM) was on the 6–9 O'clock position, a small hole in the zona pellucida was opened (**Figure 2B**) on the 3 O'clock position by one laser pulse with the ZILOS-tk™ laser system (Hamilton Thorn Bioscience Inc., MA, USA). A 20 μm polished biopsy pipette (Sunlight Medical, Jacksonville, FL, USA) was inserted to inside zona pellucida through the hole and a few trophectoderm cells on the 12–2 O'clock position were aspired into biopsy pipette (**Figure 2B**). After biopsy pipette was pulled out of the zona (**Figure 2C**), biopsy pipette together with blastocyst was moved to the top the holding pipette (**Figure 2D**), and the biopsy pipette was pull down against the holding pipette so that the cells inside the biopsy pipette were completely separated from blastocyst proper (**Figure 2E**).

After biopsy, biopsied cells were washed individually, transferred to PCR tubes, and stored at −20°C freezer until processing for PGT-A by commercial genetic testing company. Blastocysts were individually cryopreserved by vitrification for later frozen embryo transfer (FET). Blastocysts were classified as abnormal if they

#### **Figure 1.**

*Procedures for two-step blastocyst biopsy. A blastocyst with some trophectoderm cells being hatched from the hole in the zona pellucida opened on day 3 and the blastocyst is held to a proper position for biopsy (A). After a few trophectoderm cells are aspirated into biopsy pipette, one laser pulse is applied on upside of the cells (B) and another lase pulse is applied to the bottom side of cells (C) during mechanical pulling. Extra laser pulses may be necessary during pulling until the cells are completely isolated from blastocyst proper (D).*

#### **Figure 2.**

*Procedures for one-step blastocyst biopsy. A blastocyst is held to a proper position for biopsy and a small hole is opened in the zona by a laser pulse at 3 O'clock position (A). A biopsy pipette is inserted into the zona through the hole and a few trophectoderm cells are aspirated into biopsy pipette after blastocyst is collapsed or during collapsing from 12 to 2 O'clock position (B). Biopsy pipette is pull out of the zona (C) and the biopsy pipette together with blastocyst is moved to the top front of holding pipette (D). Biopsy pipette is pull down against the holding pipette to cut the cell connection between blastocyst proper and aspirated cells at the tip of biopsy pipette (E).*

had any chromosomal error(s). The abnormal samples were further divided into aneuploidy if they had gain and/or missing of a chromosome(s) and segmental abnormal if they had only deletion and/or duplication in a chromosome(s).

**79**

number of transfers.

**3. Results**

**2.6 Statistical analysis**

also similar between two groups.

between two groups (63.4 vs. 64.0%).

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

Blastocysts were vitrified using a vitrification device (Cryotop, Vitristraw or Mini straw) and kit (Fujifilm-Irvine Scientific, Irvine, CA, USA). Both equilibration solution and vitrification solution were warmed in original vials at 37°C for at least 30 min before use. Briefly, collapsed blastocysts were equilibrated in 100 μl drop (without oil cover) of equilibration solution for 2 min, and then 45 seconds in 100 μl drop (without oil cover) of vitrification solution (both steps were performed on a 37°C warming stage) before loading to vitrification device. All blastocysts were vitrified individually and then stored in liquid nitrogen until warming for FET. For warming, blastocysts were exposed to a thawing solution (Fujifilm-Irvine warming kit) at 37°C for 1 min, transferred to a dilution solution for 3 min and finally to a washing solution for 10 min with a solution change after 5 min at room temperature. After completion of the warming process, zona pellucida in the blastocysts were further cut by laser pulses to open 1/4–1/5 (2D image size) of zona pellucida and then cultured in Global™Total medium for 2–4 h before transfer. For preparation of the transfer, patients received estradiol (Estrace, Warner Chilcott, NJ, USA) orally or vaginally, and estradiol patch (Estradiol Transdermal System, Noven Pharmaceuticals, NJ, USA) every three days, as well as progesterone that was administered on 15th day of estradiol treatment. Blastocysts were transferred on the sixth or seventh day of progesterone administered, and progesterone was continued daily until the first serum β-hCG test two weeks after transfer. Ongoing pregnancy was supported by continued estradiol and progesterone until 11 weeks of pregnancy. Pregnancy was initially confirmed 14 days after embryo transfer by a serum β-hCG assay. Four weeks after embryo transfer, when a gestational sac and a heartbeat appeared, the patient was diagnosed as having a clinical pregnancy. Live birth rates were calculated based on the number of live birth and

Interval data was analyzed by one-way analysis of ANOVA. The differences between groups were compared with chi square test. If the P value was less 0.05, the

To examine whether day 3 zona hole opening by laser pulse affected embryo development, blastocyst development between embryos with or without this procedure were compared. As shown in **Table 1**, similar blastocyst development rates (64.7 vs. 64.3%) were observed between two groups. Other parameters, such as egg donor's ages (26.5 ± 3.0 vs. 25.6 ± 2.6), and fertilization rates (86.4 vs. 88.8%) were

As shown in **Table 2**, after biopsy, the proportions of samples without tested results due to low quantity of DNA or no DNA in the samples were similar between two biopsy methods (3.6 vs. 4.4%), resulting in 96.4% of the samples biopsied with one-step method and 95.6% of the samples biopsied with two-step method were successfully amplified. It was found that euploid blastocyst rates were similar

Chromosome abnormalities include whole chromosome aneuploidies (extra and/or missing chromosomes), and segmental chromosome abnormalities, such as chromosome deletion and duplication. As shown in **Table 2**, no differences were

difference was considered to be statistically significant.

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

**2.5 Blastocyst vitrification, warming and transfer**

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

#### **2.5 Blastocyst vitrification, warming and transfer**

Blastocysts were vitrified using a vitrification device (Cryotop, Vitristraw or Mini straw) and kit (Fujifilm-Irvine Scientific, Irvine, CA, USA). Both equilibration solution and vitrification solution were warmed in original vials at 37°C for at least 30 min before use. Briefly, collapsed blastocysts were equilibrated in 100 μl drop (without oil cover) of equilibration solution for 2 min, and then 45 seconds in 100 μl drop (without oil cover) of vitrification solution (both steps were performed on a 37°C warming stage) before loading to vitrification device. All blastocysts were vitrified individually and then stored in liquid nitrogen until warming for FET.

For warming, blastocysts were exposed to a thawing solution (Fujifilm-Irvine warming kit) at 37°C for 1 min, transferred to a dilution solution for 3 min and finally to a washing solution for 10 min with a solution change after 5 min at room temperature. After completion of the warming process, zona pellucida in the blastocysts were further cut by laser pulses to open 1/4–1/5 (2D image size) of zona pellucida and then cultured in Global™Total medium for 2–4 h before transfer.

For preparation of the transfer, patients received estradiol (Estrace, Warner Chilcott, NJ, USA) orally or vaginally, and estradiol patch (Estradiol Transdermal System, Noven Pharmaceuticals, NJ, USA) every three days, as well as progesterone that was administered on 15th day of estradiol treatment. Blastocysts were transferred on the sixth or seventh day of progesterone administered, and progesterone was continued daily until the first serum β-hCG test two weeks after transfer. Ongoing pregnancy was supported by continued estradiol and progesterone until 11 weeks of pregnancy. Pregnancy was initially confirmed 14 days after embryo transfer by a serum β-hCG assay. Four weeks after embryo transfer, when a gestational sac and a heartbeat appeared, the patient was diagnosed as having a clinical pregnancy. Live birth rates were calculated based on the number of live birth and number of transfers.

#### **2.6 Statistical analysis**

Interval data was analyzed by one-way analysis of ANOVA. The differences between groups were compared with chi square test. If the P value was less 0.05, the difference was considered to be statistically significant.

#### **3. Results**

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

had any chromosomal error(s). The abnormal samples were further divided into aneuploidy if they had gain and/or missing of a chromosome(s) and segmental abnormal if they had only deletion and/or duplication in a chromosome(s).

*Procedures for one-step blastocyst biopsy. A blastocyst is held to a proper position for biopsy and a small hole is opened in the zona by a laser pulse at 3 O'clock position (A). A biopsy pipette is inserted into the zona through the hole and a few trophectoderm cells are aspirated into biopsy pipette after blastocyst is collapsed or during collapsing from 12 to 2 O'clock position (B). Biopsy pipette is pull out of the zona (C) and the biopsy pipette together with blastocyst is moved to the top front of holding pipette (D). Biopsy pipette is pull down against the holding pipette to cut the cell connection between blastocyst proper and aspirated cells at the tip of biopsy* 

**78**

**Figure 2.**

*pipette (E).*

To examine whether day 3 zona hole opening by laser pulse affected embryo development, blastocyst development between embryos with or without this procedure were compared. As shown in **Table 1**, similar blastocyst development rates (64.7 vs. 64.3%) were observed between two groups. Other parameters, such as egg donor's ages (26.5 ± 3.0 vs. 25.6 ± 2.6), and fertilization rates (86.4 vs. 88.8%) were also similar between two groups.

As shown in **Table 2**, after biopsy, the proportions of samples without tested results due to low quantity of DNA or no DNA in the samples were similar between two biopsy methods (3.6 vs. 4.4%), resulting in 96.4% of the samples biopsied with one-step method and 95.6% of the samples biopsied with two-step method were successfully amplified. It was found that euploid blastocyst rates were similar between two groups (63.4 vs. 64.0%).

Chromosome abnormalities include whole chromosome aneuploidies (extra and/or missing chromosomes), and segmental chromosome abnormalities, such as chromosome deletion and duplication. As shown in **Table 2**, no differences were

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


#### **Table 1.**

*Development of human embryos with or without laser zona hole opening that was performed on cleavage stage embryos at day 3.*


#### **Table 2.**

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

#### **Figure 3.**

*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 chromosomes.*

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

**81**

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