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1.  On-Site Array CGH Applications in Clinical In Vitro Fertilization: Reproductive Outcomes and Impact on Cryopreservation of Non-transferred Human Embryos 
Background: IVF pregnancy rates have trended upward although gains have been accompanied by unwelcome increases in pre-term delivery and multiple gestation. These adverse outcomes happen because multiple embryos are typically transferred during IVF. Integrating newer molecular cytogenetic techniques with IVF can optimize selection of a single embryo for transfer. Methods: The SurePlex DNA amplification system (BlueGnome Ltd; Cambridge, UK) was used on-site for whole genome amplification of human blastocyst trophectoderm (TE) cells obtained by biopsy. IVF patients (initial cycle, age <35, no prior miscarriage, normal karyotype) were prospectively randomized into two groups: In Group 1, embryos were selected on the basis of morphology and comprehensive chromosomal screening via array comparative genomic hybridization (aCGH) from d5 TE biopsy, while Group 2 embryos were assessed by morphology only. All patients underwent a single fresh blastocyst transfer on d6. For embryos in the aCGH group, only one euploid blastocyst was selected for transfer and surplus euploid blastocysts were vitrified. In the non-aCGH (control) group, a single blastocyst was selected for fresh transfer based on appearance only, with vitrification of any surplus blastocysts with satisfactory morphology. Results: Aneuploidy was identified in 191/425 of Group 1 balstocysts (44.9%). Control embryos (n=389) were assessed by microscopy only. A higher clinical pregnancy rate was observed in Group 1 patients compared to the control group (70.9 vs. 45.8%; p = 0.017). Only 64 (28.3%) surplus euploid embryos were frozen in Group 1 while 157 (40.4%) blastocysts were cryopreserved for Group 2 (p=0.017). Conclusion: These data underscore the intrinsic imprecision of IVF when conventional morphology is used alone to select embryos for transfer. Embryos evaluated with aCGH implant with greater efficiency and achieve clinical pregnancy more often than those selected without aCGH. Patients should be advised that aCGH screening may reduce the number of surplus embryos for cryopreservation.
PMCID: PMC3635430
2.  Selection of euploid blastocysts for cryopreservation with array comparative genomic hybridization (aCGH) results in increased implantation rates in subsequent frozen and thawed embryo transfer cycles 
In assisted reproductive treatments, embryos remaining after fresh embryo transfer are usually selected for cryopreservation based on traditional morphology assessment. Our previous report has demonstrated that array comparative genomic hybridization (aCGH) screening for IVF patients with good prognosis significantly improves clinical and ongoing pregnancy rates in fresh embryo transfer cycles. The current study further investigates the efficiency of applying aCGH in the selection of euploid embryos for cryopreservation as related to pregnancy and implantation outcomes in subsequent frozen embryo transfer (FET) cycles.
First-time IVF patients with good prognosis undergoing fresh single embryo transfer and having at least one remaining blastocyst for cryopreservation were prospectively randomized into two groups: 1) Group A patients had embryos assessed by morphology first and then by aCGH screening of trophectoderm cells and 2) Group B patients had embryos evaluated by morphology alone. All patients had at least one blastocyst available for cryopreservation after fresh embryo transfer. There were 15 patients in Group A and 23 patients in Group B who failed to conceive after fresh embryo transfer and completed the FET cycles. Blastocyst survival and implantation rates were compared between the two groups.
There were no significant differences in blastocyst survival rates between Group A and Group B (90.9% vs. 91.3%, respectively; p >0.05). However, a significantly higher implantation rate was observed in the morphology assessment plus aCGH screening group compared to the morphology assessment alone group (65.0% vs. 33.3%, respectively; p = 0.038). There was no miscarriage observed in Group A while a 16.7% miscarriage rate was recorded in Group B (0% vs. 16.7%, respectively; p >0.05).
While aCGH screening has been recently applied to select euploid blastocysts for fresh transfer in young, low-risk IVF patients, this is the first prospective study on the impact of aCGH specifically on blastocyst survival and implantation outcomes in the subsequent FET cycles of IVF patients with good prognosis. The present study demonstrates that aCGH screening of blastocysts prior to cryopreservation significantly improves implantation rates and may reduce the risk of miscarriage in subsequent FET cycles. Further randomized clinical studies with a larger sample size are needed to validate these preliminary findings.
PMCID: PMC3766007  PMID: 23937723
aCGH; Trophectoderm biopsy; Cryopreservation; Implantation
3.  Selection of single blastocysts for fresh transfer via standard morphology assessment alone and with array CGH for good prognosis IVF patients: results from a randomized pilot study 
Single embryo transfer (SET) remains underutilized as a strategy to reduce multiple gestation risk in IVF, and its overall lower pregnancy rate underscores the need for improved techniques to select one embryo for fresh transfer. This study explored use of comprehensive chromosomal screening by array CGH (aCGH) to provide this advantage and improve pregnancy rate from SET.
First-time IVF patients with a good prognosis (age <35, no prior miscarriage) and normal karyotype seeking elective SET were prospectively randomized into two groups: In Group A, embryos were selected on the basis of morphology and comprehensive chromosomal screening via aCGH (from d5 trophectoderm biopsy) while Group B embryos were assessed by morphology only. All patients had a single fresh blastocyst transferred on d6. Laboratory parameters and clinical pregnancy rates were compared between the two groups.
For patients in Group A (n = 55), 425 blastocysts were biopsied and analyzed via aCGH (7.7 blastocysts/patient). Aneuploidy was detected in 191/425 (44.9%) of blastocysts in this group. For patients in Group B (n = 48), 389 blastocysts were microscopically examined (8.1 blastocysts/patient). Clinical pregnancy rate was significantly higher in the morphology + aCGH group compared to the morphology-only group (70.9 and 45.8%, respectively; p = 0.017); ongoing pregnancy rate for Groups A and B were 69.1 vs. 41.7%, respectively (p = 0.009). There were no twin pregnancies.
Although aCGH followed by frozen embryo transfer has been used to screen at risk embryos (e.g., known parental chromosomal translocation or history of recurrent pregnancy loss), this is the first description of aCGH fully integrated with a clinical IVF program to select single blastocysts for fresh SET in good prognosis patients. The observed aneuploidy rate (44.9%) among biopsied blastocysts highlights the inherent imprecision of SET when conventional morphology is used alone. Embryos randomized to the aCGH group implanted with greater efficiency, resulted in clinical pregnancy more often, and yielded a lower miscarriage rate than those selected without aCGH. Additional studies are needed to verify our pilot data and confirm a role for on-site, rapid aCGH for IVF patients contemplating fresh SET.
PMCID: PMC3403960  PMID: 22551456
4.  Randomized comparison of next-generation sequencing and array comparative genomic hybridization for preimplantation genetic screening: a pilot study 
BMC Medical Genomics  2015;8:30.
Recent advances in next-generation sequencing (NGS) have provided new methods for preimplantation genetic screening (PGS) of human embryos from in vitro fertilization (IVF) cycles. However, there is still limited information about clinical applications of NGS in IVF and PGS (IVF-PGS) treatments. The present study aimed to investigate the effects of NGS screening on clinical pregnancy and implantation outcomes for PGS patients in comparison to array comparative genomic hybridization (aCGH) screening.
This study was performed in two phases. Phase I study evaluated the accuracy of NGS for aneuploidy screening in comparison to aCGH. Whole-genome amplification (WGA) products (n = 164) derived from previous IVF-PGS cycles (n = 38) were retrospectively analyzed with NGS. The NGS results were then compared with those of aCGH. Phase II study further compared clinical pregnancy and implantation outcomes between NGS and aCGH for IVF-PGS patients. A total of 172 patients at mean age 35.2 ± 3.5 years were randomized into two groups: 1) NGS (Group A): patients (n = 86) had embryos screened with NGS and 2) aCGH (Group B): patients (n = 86) had embryos screened with aCGH. For both groups, blastocysts were vitrified after trophectoderm biopsy. One to two euploid blastocysts were thawed and transferred to individual patients primarily based on the PGS results. Ongoing pregnancy and implantation rates were compared between the two study groups.
NGS detected all types of aneuploidies of human blastocysts accurately and provided a 100 % 24-chromosome diagnosis consistency with the highly validated aCGH method. Moreover, NGS screening identified euploid blastocysts for transfer and resulted in similarly high ongoing pregnancy rates for PGS patients compared to aCGH screening (74.7 % vs. 69.2 %, respectively, p >0.05). The observed implantation rates were also comparable between the NGS and aCGH groups (70.5 % vs. 66.2 %, respectively, p >0.05).
While NGS screening has been recently introduced to assist IVF patients, this is the first randomized clinical study on the efficiency of NGS for preimplantation genetic screening in comparison to aCGH. With the observed high accuracy of 24-chromosome diagnosis and the resulting high ongoing pregnancy and implantation rates, NGS has demonstrated an efficient, robust high-throughput technology for PGS.
PMCID: PMC4477308  PMID: 26100406
NGS; aCGH; PGS; Aneuploidy screening; Ongoing pregnancy; Implantation
5.  Two different microarray technologies for preimplantation genetic diagnosis and screening, due to reciprocal translocation imbalances, demonstrate equivalent euploidy and clinical pregnancy rates 
To compare single nucleotide polymorphism (SNP) and comparative genomic hybridization (aCGH) microarray platforms to evaluate embryos for parental translocation imbalances and aneuploidy.
A retrospective review of preimplantation genetic diagnosis and screening (PGD/PGS) results of 498 embryos from 63 couples undergoing 75 in vitro fertilization cycles due to parental carriers of a reciprocal translocation.
There was no significant difference between SNP and aCGH microarrays when comparing the prevalence of embryos that were euploid with no translocation imbalance, euploidy with a parental translocation imbalance or aneuploid with or without the parental chromosome imbalance. The clinical pregnancy rates were also equivalent for SNP (60 %) versus aCGH (65 %) microarrays. Of 498 diagnosed embryos, 45 % (226/498) were chromosomally normal without translocation errors or aneuploidy, 22 % (112/498) were euploid but had a parentally derived unbalanced chromosomal segregant, 8 % (42/498) harbored both a translocation imbalance and aneuploidy and 24 % (118/498) of embryos were genetically balanced for the parental reciprocal translocation but were aneuploid for other chromosomes. The overall clinical pregnancy rate per IVF cycle following SNP or aCGH microarray analysis was 61 % and was higher if the biopsy was done on blastocysts (65 %) versus cleavage stage embryos (59 %), although not statistically significant.
SNP or aCGH microarray technologies demonstrate equivalent clinical findings that maximize the pregnancy potential in patients with known parental reciprocal chromosomal translocations.
PMCID: PMC4096875  PMID: 24771116
Translocation; PGD; PGS; Microarray; IVF; Reciprocal
6.  Increasing Live Birth Rate by Preimplantation Genetic Screening of Pooled Polar Bodies Using Array Comparative Genomic Hybridization 
PLoS ONE  2015;10(5):e0128317.
Meiotic errors during oocyte maturation are considered the major contributors to embryonic aneuploidy and failures in human IVF treatment. Various technologies have been developed to screen polar bodies, blastomeres and trophectoderm cells for chromosomal aberrations. Array-CGH analysis using bacterial artificial chromosome (BAC) arrays is widely applied for preimplantation genetic diagnosis (PGD) using single cells. Recently, an increase in the pregnancy rate has been demonstrated using array-CGH to evaluate trophectoderm cells. However, in some countries, the analysis of embryonic cells is restricted by law. Therefore, we used BAC array-CGH to assess the impact of polar body analysis on the live birth rate. A disadvantage of polar body aneuploidy screening is the necessity of the analysis of both the first and second polar bodies, resulting in increases in costs for the patient and complex data interpretation. Aneuploidy screening results may sometimes be ambiguous if the first and second polar bodies show reciprocal chromosomal aberrations. To overcome this disadvantage, we tested a strategy involving the pooling of DNA from both polar bodies before DNA amplification. We retrospectively studied 351 patients, of whom 111 underwent polar body array-CGH before embryo transfer. In the group receiving pooled polar body array-CGH (aCGH) analysis, 110 embryos were transferred, and 29 babies were born, corresponding to live birth rates of 26.4% per embryo and 35.7% per patient. In contrast, in the control group, the IVF treatment was performed without preimplantation genetic screening (PGS). For this group, 403 embryos were transferred, and 60 babies were born, resulting in live birth rates of 14.9% per embryo and 22.7% per patient. In conclusion, our data show that in the aCGH group, the use of aneuploidy screening resulted in a significantly higher live birth rate compared with the control group, supporting the benefit of PGS for IVF couples in addition to the suitability and effectiveness of our polar body pooling strategy.
PMCID: PMC4449032  PMID: 26024488
7.  Comparing thaw survival, implantation and live birth rates from cryopreserved zygotes, embryos and blastocysts 
Most in vitro fertilization (IVF) programs employ embryo cryopreservation to enhance pregnancies from a single ovarian stimulation. More embryos are created, some of which are not transferred to the uterus immediately, generating a need for improved cryopreservation protocols. One protocol may involve growing embryos to a further stage of development, allowing only embryos with proven developmental capabilities to be cryopreserved. Here we examined thaw survival, implantation and live birth rates of embryos cryopreserved at different stages.
We examined thaw survival, implantation and live birth rates of embryos cryopreserved at the zygote, day 3 (D3) embryos or blastocyst stage.
This is a retrospective study from a single academic IVF program.
A retrospective study of all patients who had frozen embryos transferred to their uteri from year 2002 to 2008 at a single academic IVF program was conducted.
Analysis of variance followed by Fisher's Exact Test was performed to compare the survival after thaw, implantation and live birth rates between the three groups.
One thousand nine hundred and ninety-one zygotes, 2880 D3 embryos and 503 blastocysts were frozen using a slow freeze technique, thawed and transferred. Significantly more D3 embryos and blastocysts survived the thawing process compared to zygotes and significantly higher implantation rate per number of thawed blastocysts was achieved than that for zygotes. Live birth rates were similar between the three groups.
Growing embryos to blastocyst stage prior to cryopreservation is associated with fewer frozen embryos but does not appear compromise patients’ chance of achieving pregnancy
PMCID: PMC3136065  PMID: 21772736
Blastocyst; embryo cryopreservation; frozen embryo transfer; implantation; in vitro fertilization; slow-freeze; zygote
8.  Selection of competent blastocysts for transfer by combining time-lapse monitoring and array CGH testing for patients undergoing preimplantation genetic screening: a prospective study with sibling oocytes 
BMC Medical Genomics  2014;7:38.
Recent advances in time-lapse monitoring in IVF treatment have provided new morphokinetic markers for embryonic competence. However, there is still very limited information about the relationship between morphokinetic parameters, chromosomal compositions and implantation potential. Accordingly, this study aimed at investigating the effects of selecting competent blastocysts for transfer by combining time-lapse monitoring and array CGH testing on pregnancy and implantation outcomes for patients undergoing preimplantation genetic screening (PGS).
A total of 1163 metaphase II (MII) oocytes were retrieved from 138 PGS patients at a mean age of 36.6 ± 2.4 years. These sibling MII oocytes were then randomized into two groups after ICSI: 1) Group A, oocytes (n = 582) were cultured in the time-lapse system and 2) Group B, oocytes (n = 581) were cultured in the conventional incubator. For both groups, whole genomic amplification and array CGH testing were performed after trophectoderm biopsy on day 5. One to two euploid blastocysts within the most predictive morphokinetic parameters (Group A) or with the best morphological grade available (Group B) were selected for transfer to individual patients on day 6. Ongoing pregnancy and implantation rates were compared between the two groups.
There were significant differences in clinical pregnancy rates between Group A and Group B (71.1% vs. 45.9%, respectively, p = 0.037). The observed implantation rate per embryo transfer significantly increased in Group A compared to Group B (66.2% vs. 42.4%, respectively, p = 0.011). Moreover, a significant increase in ongoing pregnancy rates was also observed in Group A compared to Group B (68.9% vs. 40.5%. respectively, p = 0.019). However, there was no significant difference in miscarriage rate between the time-lapse system and the conventional incubator (3.1% vs. 11.8%, respectively, p = 0.273).
This is the first prospective investigation using sibling oocytes to evaluate the efficiency of selecting competent blastocysts for transfer by combining time-lapse monitoring and array CGH testing for PGS patients. Our data clearly demonstrate that the combination of these two advanced technologies to select competent blastocysts for transfer results in improved implantation and ongoing pregnancy rates for PGS patients.
PMCID: PMC4077552  PMID: 24954518
Time-lapse monitoring; Array CGH; PGS; Ploidy; Implantation; Miscarriage
9.  Comparative Genomic Hybridization Selection of Blastocysts for Repeated Implantation Failure Treatment: A Pilot Study 
BioMed Research International  2014;2014:457913.
The aim of this study is to determine if the use of preimplantation genetic screening (PGS) by array comparative genomic hybridization (array CGH) and transfer of a single euploid blastocyst in patients with repeated implantation failure (RIF) can improve clinical results. Three patient groups are compared: 43 couples with RIF for whom embryos were selected by array CGH (group RIF-PGS), 33 couples with the same history for whom array CGH was not performed (group RIF NO PGS), and 45 good prognosis infertile couples with array CGH selected embryos (group NO RIF PGS). A single euploid blastocyst was transferred in groups RIF-PGS and NO RIF PGS. Array CGH was not performed in group RIF NO PGS in which 1-2 blastocysts were transferred. One monoembryonic sac with heartbeat was found in 28 patients of group RIF PGS and 31 patients of group NO RIF PGS showing similar clinical pregnancy and implantation rates (68.3% and 70.5%, resp.). In contrast, an embryonic sac with heartbeat was only detected in 7 (21.2%) patients of group RIF NO PGS. In conclusion, PGS by array CGH with single euploid blastocyst transfer appears to be a successful strategy for patients with multiple failed IVF attempts.
PMCID: PMC3980987  PMID: 24779011
10.  Ovarian hyperstimulation syndrome and prophylactic human embryo cryopreservation: analysis of reproductive outcome following thawed embryo transfer 
To review utilisation of elective embryo cryopreservation in the expectant management of patients at risk for developing ovarian hyperstimulation syndrome (OHSS), and report on reproductive outcome following transfer of thawed embryos.
Materials and methods
Medical records were reviewed for patients undergoing IVF from 2000–2008 to identify cases at risk for OHSS where cryopreservation was electively performed on all embryos at the 2 pn stage. Patient age, total number of oocytes retrieved, number of 2 pn embryos cryopreserved, interval between retrieval and thaw/transfer, number (and developmental stage) of embryos transferred (ET), and delivery rate after IVF were recorded for all patients.
From a total of 2892 IVF cycles undertaken during the study period, 51 IVF cases (1.8%) were noted where follicle number exceeded 20 and pelvic fluid collection was present. Elective embryo freeze was performed as OHSS prophylaxis in each instance. Mean (± SD) age of these patients was 32 ± 3.8 yrs. Average number of oocytes retrieved in this group was 23 ± 8.7, which after fertilisation yielded an average of 14 ± 5.7 embryos cryopreserved per patient. Thaw and ET was performed an average of 115 ± 65 d (range 30–377 d) after oocyte retrieval with a mean of 2 ± 0.6 embryos transferred. Grow-out to blastocyst stage was achieved in 88.2% of cases. Delivery/livebirth rate was 33.3% per initiated cycle and 43.6% per transfer. Non-transferred blastocysts remained in cryostorage for 24 of 51 patients (46.1%) after ET, with an average of 3 ± 3 blastocysts refrozen per patient.
OHSS prophylaxis was used in 1.8% of IVF cycles at this institution; no serious OHSS complications were encountered during the study period. Management based on elective 2 pn embryo cryopreservation with subsequent thaw and grow-out to blastocyst stage for transfer did not appear to compromise embryo viability or overall reproductive outcome. For these patients, immediate elective embryo cryopreservation and delay of ET by as little as 30 d allowed for satisfactory conclusion of the IVF sequence, yielding a livebirth-delivery rate (per ET) >40%.
PMCID: PMC2585559  PMID: 19014420
11.  Assessment of aneuploidy formation in human blastocysts resulting from cryopreserved donor eggs 
Increased embryo implantation rates were reported after transfer of euploid embryos selected by preimplantation genetic screening (PGS). Egg cryopreservation by vitrification has become one of the most important assisted human reproduction technologies. Although reports indicate that development and implantation of human embryos derived from frozen donor eggs are comparative to fresh eggs, it is still unknown whether egg vitrification increases chromosomal abnormalities in eggs, which in turn causes formation of embryonic aneuploidy. Therefore, in this study, we evaluated the aneuploidy formation in the blastocysts derived from frozen donor eggs and also evaluated the efficiency of egg vitrification as an advanced technology for egg cryopreservation.
In this study, donated human eggs from young women were cryopreserved by vitrification and PGS was performed in the resulted blastocysts by DNA microarray. A total of 764 frozen eggs from 75 egg thawing cycles were warmed and 38 blastocysts were biopsied for PGS before embryo transfer. A 97.1% of egg survival rate was obtained and 59.1% of embryos developed to blastocyst stage. After biopsy and PGS, it was found that 84.2% of blastocysts were euploid and 15.8% were aneuploid. Aneuploidy rates varied among donors. Transfers of blastocysts without PGS resulted in higher clinical pregnancy and implantation rates as compared with transfer of blastocysts with PGS.
Although the overall aneuploidy rate was low in the blastocysts derived from frozen donor eggs, high aneuploidy rates were observed in the embryos resulting from some donated eggs. Clinical pregnancy rate was not improved by PGS of embryos resulting from donor eggs, indicating that PGS may not be necessary for embryos derived from donor eggs in most cases.
Electronic supplementary material
The online version of this article (doi:10.1186/s13039-015-0117-8) contains supplementary material, which is available to authorized users.
PMCID: PMC4339107  PMID: 25713597
Aneuploidy; Egg freezing; Donor eggs; Implantation
12.  Live birth following serial vitrification of embryos and PGD for fragile X syndrome in a patient with the premutation and decreased ovarian reserve 
To present a live birth resulting from serial vitrification of embryos and pre-implantation genetic diagnosis (PGD).
A 31-year-old with primary infertility, fragile-X premutation, and decreased ovarian reserve (DOR) (baseline FSH level 33 IU/L), presented after failing to stimulate to follicle diameters >10 mm with three cycles of invitro fertilization (IVF). After counseling, the couple opted for serial in-vitro maturation (IVM), embryo vitrification, and genetic testing using array comparative genomic hybridization (aCGH) and PGD. Embryos were vitrified 2 days after intra-cytoplasmic sperm injection (ICSI). Thawed embryos were biopsied on day-three and transferred on day-five.
The couple underwent 20 cycles of assisted reproductive technology. A total of 23 in-vivo mature and five immature oocytes were retrieved, of which one matured in-vitro. Of 24 embryos, 17/24 (71 %) developed to day two and 11/24 (46 %) survived to blastocyst stage with a biopsy result available. Four blastocysts had normal PGD and aCGH results. Both single embryo transfers resulted in a successful implantation, one a blighted ovum and the other in a live birth.
Young patients with DOR have potential for live birth as long as oocytes can be obtained and embryos created. Serial vitrification may be the mechanism of choice in these patients when PGD is needed.
PMCID: PMC3879937  PMID: 24062195
In-vitro maturation (IVM); Embryo vitrification; Diminished Ovarian Reserve (DOR); Fragile X Syndrome (FXS); Preimplantation Genetic Diagnosis (PGD); Array Comparative Genomic Hybridization (aCGH)
13.  An update of preimplantation genetic diagnosis in gene diseases, chromosomal translocation, and aneuploidy screening 
Preimplantation genetic diagnosis (PGD) is gradually widely used in prevention of gene diseases and chromosomal abnormalities. Much improvement has been achieved in biopsy technique and molecular diagnosis. Blastocyst biopsy can increase diagnostic accuracy and reduce allele dropout. It is cost-effective and currently plays an important role. Whole genome amplification permits subsequent individual detection of multiple gene loci and screening all 23 pairs of chromosomes. For PGD of chromosomal translocation, fluorescence in-situ hybridization (FISH) is traditionally used, but with technical difficulty. Array comparative genomic hybridization (CGH) can detect translocation and 23 pairs of chromosomes that may replace FISH. Single nucleotide polymorphisms array with haplotyping can further distinguish between normal chromosomes and balanced translocation. PGD may shorten time to conceive and reduce miscarriage for patients with chromosomal translocation. PGD has a potential value for mitochondrial diseases. Preimplantation genetic haplotyping has been applied for unknown mutation sites of single gene disease. Preimplantation genetic screening (PGS) using limited FISH probes in the cleavage-stage embryo did not increase live birth rates for patients with advanced maternal age, unexplained recurrent abortions, and repeated implantation failure. Polar body and blastocyst biopsy may circumvent the problem of mosaicism. PGS using blastocyst biopsy and array CGH is encouraging and merit further studies. Cryopreservation of biopsied blastocysts instead of fresh transfer permits sufficient time for transportation and genetic analysis. Cryopreservation of embryos may avoid ovarian hyperstimulation syndrome and possible suboptimal endometrium.
PMCID: PMC3283069  PMID: 22384431
Array Comparative Genomic Hybridization; Preimplantation Genetic Diagnosis; Preimplantation Genetic Screening; Single Nucleotide Polymorphisms Array; Vitrification; Whole Genome Amplification; Human
14.  Vitrification of mouse embryos at 2-cell, 4-cell and 8-cell stages by cryotop method 
The objective of this study was to investigate the effects of vitrification on the preimplantation developmental competence of mouse 2-cell, 4-cell and 8-cell stage embryos.
Mouse 2-cell, 4-cell and 8-cell stage embryos were cryopreserved using the cryotop vitrification method and subsequently warmed on a later date. The embryos were then assessed by their morphology, blastocyst formation and hatching rates. Additionally, trophectoderm (TE) and inner cell mass (ICM) cell numbers were compared in hatched blastocysts from the control and experimental groups.
Vitrified embryos at the 2-cell, 4-cell and 8-cell stages appeared morphologically normal after warming. The overall survival rate of vitrified embryos at various stages after warming was 96.7% and there were no significant differences among 2-cell stage (96.0%), 4-cell stage (96.8%) and 8-cell stage (97.1%) embryos (P > 0.05). The blastocyst formation rate (69.4%) and hatching rate (52.6%) of vitrified 2-cell embryos were significantly lower than that from the control group and vitrified 8-cell embryos (P < 0.05). In the vitrified 4-cell embryo group, the blastocyst formation rate (90.3%) was similar to the 8-cell group (91.2%), but the hatching rate (60.0%) was significantly lower than that of the non-vitrified control ( 84.1%) and vitrified 8-cell embryo (78.4%) groups (P < 0.05). When further development to the fully hatched blastocyst stage was compared, hatched blastocysts derived from vitrified 2-cell, 4-cell and 8-cell embryos had significantly lower cell counts both in the ICM and TE, as compared to fresh blastocysts (P < 0.05). Among the vitrified 2-cell, 4-cell and 8-cell embryo groups, there were no significant differences in the cell counts of ICM and TE (P > 0.05).
Although cryotop vitrification was suitable for the cryopreservation of mouse embryos from the 2-cell stage, 4-cell stage and 8-cell stage without significant loss of survival, vitrification had an adverse effect on the development of 2-cell embryos. Mouse embryos at the 8-cell stage had the best tolerance for vitrification and would yield the highest level of post-vitrification developmental competence among early cleavage stage embryos. Nevertheless, it is unclear how these findings can be extrapolated to human embryos.
PMCID: PMC2799565  PMID: 19967554
Vitrification; Cleavage; Embryos; Development; Cryopreservation; Mouse
15.  A greater number of euploid blastocysts in a given cohort predicts excellent outcomes in single embryo transfer cycles 
This multicentered retrospective study analyzed whether the quantity of euploid blastocysts in a given cohort after comprehensive chromosomal screening can be used to identify candidates for single embryo transfer.
Blastocysts from 437 patients underwent trophectoderm biopsy followed by array comparative genomic hybridization. Embryos were then selected for single or double embryo transfer. The number of euploid blastocysts produced and transferred for each patient was recorded, as was clinical pregnancy rate and multiple gestation rate.
In patients with ≤3 euploid blastocysts, clinical pregnancy rate was higher in double, compared to single embryo transfers. However, in patients with ≥4 euploid blastocysts, clinical pregnancy rate was not reduced with single embryo transfer was performed, whereas the multiple gestation rate was greatly reduced.
Size of the euploid embryo cohort is a marker for success in single embryo transfer cycles. Patients who produce at least four euploid blastocysts are outstanding candidates for single embryo transer.
PMCID: PMC4048376  PMID: 24659020
Single embryo transfer (SET); Preimplantation Genetic Screening (PGS); Trophectoderm biopsy; Array comparative genomic hybridization (array-CGH); Assisted reproductive technology (ART)
16.  Comparison of clinical outcomes between fresh embryo transfers and frozen-thawed embryo transfers 
Background: Advances in embryo culture technology and cryopreservation have led to a shift in in vitro fertilization (IVF) from early fresh or frozen-thawed cleavage embryo transfer to fresh or frozen-thawed blastocyst stage transfer.
Objective: To compare the clinical outcomes of fresh embryo transfers and frozen-thawed embryo transfers.
Materials and Methods: In this retrospective case control study, patients undergoing IVF cycles from January 2012 to December 2012 were enrolled in Assisted Reproduction of Wuhan Union Hospital were enrolled. A total of 1891 cycle contains 1150 fresh embryo transfers and 741 frozen-thawed embryo transfers were studied. All data were transferred directly to SPSS 18 and analyzed.
Results: Clinical pregnancy rates of fresh cleavage-stage embryo transfers compared with fresh blastocyst transfers, frozen-thawed cleavage-stage embryo transfers, post thaw cleavage-stage extended blastocyst culture transfers and frozen-thawed blastocyst transfers were 52.7%, 35.88%, 35.29%, 47.75%, 59.8% in patients under 35 years of ages and 41.24%, 26.92%, 11.32%, 46.15%, 55.8% in patients older than 35 years old, respectively. The multiple pregnancy rates, abortion rates and ectopic pregnancy rates did not differ significantly among the five groups.
Conclusion: The clinical pregnancy rates were not different significantly between fresh cleavage-stage embryo transfers and fresh blastocyst transfers. But the clinical pregnancy rate of frozen-thawed blastocyst transfer was the highest among fresh/frozen-thawed embryo transfers.
PMCID: PMC4111889  PMID: 25071849
In vitro fertilization; Embryo transfer; Clinical outcome; Ectopic pregnancy; Endometrial receptivity
17.  New Tools for Embryo Selection: Comprehensive Chromosome Screening by Array Comparative Genomic Hybridization 
BioMed Research International  2014;2014:517125.
The objective of this study was to evaluate the usefulness of comprehensive chromosome screening (CCS) using array comparative genomic hybridization (aCGH). The study included 1420 CCS cycles for recurrent miscarriage (n = 203); repetitive implantation failure (n = 188); severe male factor (n = 116); previous trisomic pregnancy (n = 33); and advanced maternal age (n = 880). CCS was performed in cycles with fresh oocytes and embryos (n = 774); mixed cycles with fresh and vitrified oocytes (n = 320); mixed cycles with fresh and vitrified day-2 embryos (n = 235); and mixed cycles with fresh and vitrified day-3 embryos (n = 91). Day-3 embryo biopsy was performed and analyzed by aCGH followed by day-5 embryo transfer. Consistent implantation (range: 40.5–54.2%) and pregnancy rates per transfer (range: 46.0–62.9%) were obtained for all the indications and independently of the origin of the oocytes or embryos. However, a lower delivery rate per cycle was achieved in women aged over 40 years (18.1%) due to the higher percentage of aneuploid embryos (85.3%) and lower number of cycles with at least one euploid embryo available per transfer (40.3%). We concluded that aneuploidy is one of the major factors which affect embryo implantation.
PMCID: PMC4022197  PMID: 24877108
18.  Cryopreservation of Embryos, Blastocysts, and Pregnancy Rates of Blastocysts Derived from Frozen-Thawed Embryos and Frozen-Thawed Blastocysts 
Purpose: To evaluate the development of cryopreserved embryos when thawed and subsequently cultured to the blastocyst stage in comparison to transferring cryopreserved blastocysts.
Methods: In this retrospective clinical study, we have evaluated 170 cycles in patients undergoing IVF treatment for infertility. Cryopreserved embryos were thawed and were subsequently cultured and transferred at the blastocyst stage. Cryopreserved blastocysts (Day 6) were thawed and transferred immediately.
Results: Five hundred and sixty embryos and 444 blastocysts have been thawed. In the embryos group, the survival rate was 89% while in the blastocyst group the survival rate was 56%. In the embryos group the blastocyst development rate was 24.5%. The implantation rate in the embryos group was 20.6% per group blastocyst transferred compared to 5.3% in the blastocyst group.
Conclusions: The ability of cryopreserved embryos to develop to blastocysts and their implantation potential does not seem to be greatly affected by the cryopreservation procedure.
PMCID: PMC3455699  PMID: 11804424
blastocyst; cryopreservation; embryos; implantation rate; pregnancy rate
19.  Clinical effectiveness of elective single versus double embryo transfer: meta-analysis of individual patient data from randomised trials 
Objective To compare the effectiveness of elective single embryo transfer versus double embryo transfer on the outcomes of live birth, multiple live birth, miscarriage, preterm birth, term singleton birth, and low birth weight after fresh embryo transfer, and on the outcomes of cumulative live birth and multiple live birth after fresh and frozen embryo transfers.
Design One stage meta-analysis of individual patient data.
Data sources A systematic review of English and non-English articles from Medline, Embase, and the Cochrane Central Register of Controlled Trials (up to 2008). Additional studies were identified by contact with clinical experts and searches of bibliographies of all relevant primary articles. Search terms included embryo transfer, randomised controlled trial, controlled clinical trial, single embryo transfer, and double embryo transfer.
Review methods Comparisons of the clinical effectiveness of cleavage stage (day 2 or 3) elective single versus double embryo transfer after fresh or frozen in vitro fertilisation (IVF) or intracytoplasmic sperm injection (ICSI) treatments were included. Trials were included if the intervention differed only in terms of the intended number of embryos to be transferred. Trials that involved only blastocyst (day five) transfers were excluded.
Results Individual patient data were received for every patient recruited to all eight eligible trials (n=1367). A total of 683 and 684 women randomised to the single and double embryo transfer arms, respectively, were included in the analysis. Baseline characteristics in the two groups were comparable. The overall live birth rate in a fresh IVF cycle was lower after single (181/683, 27%) than double embryo transfer (285/683, 42%) (adjusted odds ratio 0.50, 95% confidence interval 0.39 to 0.63), as was the multiple birth rate (3/181 (2%) v 84/285 (29%)) (0.04, 0.01 to 0.12). An additional frozen single embryo transfer, however, resulted in a cumulative live birth rate not significantly lower than the rate after one fresh double embryo transfer (132/350 (38%) v 149/353 (42%) (0.85, 0.62 to 1.15), with a minimal cumulative risk of multiple birth (1/132 (1%) v 47/149 (32%)). The odds of a term singleton birth (that is, over 37 weeks) after elective single embryo transfer was almost five times higher than the odds after double embryo transfer (4.93, 2.98 to 8.18).
Conclusions Elective single embryo transfer results in a higher chance of delivering a term singleton live birth compared with double embryo transfer. Although this strategy yields a lower pregnancy rate than a double embryo transfer in a fresh IVF cycle, this difference is almost completely overcome by an additional frozen single embryo transfer cycle. The multiple pregnancy rate after elective single embryo transfer is comparable with that observed in spontaneous pregnancies.
PMCID: PMC3006495  PMID: 21177530
20.  Blastocyst development rate impacts outcome in cryopreserved blastocyst transfer cycles 
Fertility and sterility  2008;90(6):2138-2143.
Structured Abstract
To assess cycle outcome among day 5 and day 6 cryopreserved frozen-thawed blastocyst embryo transfers (FBET).
Retrospective cohort study.
Military-based ART center.
One hundred seventy-two non-donor, programmed cryopreserved embryo cycles.
Fully expanded blastocysts on day 5 were cryopreserved on day 5 and those achieving this state on day 6 were cryopreserved on day 6. Leuprolide acetate was given for ovulation inhibition and endometrial supplementation was by oral and vaginal estradiol. Progesterone in oil was administered and blastocyst transfer occurred in the morning of the 6th day of progesterone.
Main Outcome Measures
Implantation, pregnancy and live birth rates.
Fresh and frozen cycle characteristics were similar between groups. Day 5 FBET had significantly higher implantation rates (32.2% vs. 19.2%; p=0.01) which remained significant even when adjusting for co-variates (OR: 1.91; 95%CI: 1.00, 3.67). Live birth rates trended towards improvement after adjusting for co-variates (OR: 1.18; 95%CI: 0.61, 2.30).
Cryopreserved day 5 blastocysts have higher implantation rates and trend toward improved pregnancy outcomes compared to cryopreserved day 6 blastocysts. This suggests that embryo development rate may, in part, predict implantation and subsequent FBET outcomes although embryos not achieving the blastocyst stage until day 6 still demonstrate acceptable outcomes.
PMCID: PMC2613799  PMID: 18178191
frozen-thawed blastocyst cycle; embryo transfer; day 5 vs. day 6
21.  Predictive value of preimplantation genetic diagnosis for aneuploidy screening in repeated IVF-ET cycles among women with recurrent implantation failure 
To determine the predictive value of euploid embryos in women with recurrent implantation failure undergoing repeated IVF-ET cycles with PGD (PGD).
Cohort of IVF-PGD cycles in a tertiary care ART facility.
Materials and method(s)
Fifty-five consecutive patients with repeated implantation failure (more than three failed IVF-ET cycles) underwent two or more PGD cycles for aneuploidy testing. Mean maternal age was 37.6 ± 5.3 years. Biopsies were performed on day 3. One blastomere was removed from each pre-embryo, fixed and analyzed by multicolor and multi-probe FISH for chromosomes X and Y, 13, 15, 16, 17, 18, 21, and 22.
Forty-three of 55 patients (78%) undergoing PGD had at least one euploid embryo for transfer. Of these 31 patients (72%) also had at least one euploid embryo available for transfer with the second cycle. Of the 12 (28%) patients with no euploid embryos available for transfer with the second IVF/PGD cycle, five had a third cycle of PGD and two of these had euploid embryos available for transfer. Seventeen of the 31 patients (55%) who had euploid embryos on the second PGD cycle conceived. The ongoing pregnancy and implantation rates in patients with at least one euploid embryo were 40% and 18%, respectively. Twelve of the 55 patients (22%) had no euploid embryos available for transfer on the first PGD cycle, but on the second PGD cycle, six (50%) of these had euploid embryos for transfer. Only two pregnancies were achieved among this group of women, yielding a pregnancy rate of 17%, but both conceptions resulted in miscarriage. Of the six patients with no euploid embryos available after the second PGD cycle, four patients had a third IVF/PGD cycle, but none had euploid embryos available for transfer. Also, among women with euploid embryos available only in either the first or second PGD cycle, but not both, no ongoing pregnancy was achieved. No woman who had a PGD cycle productive of no euploid embryos had an ongoing pregnancy. Significant differences were found in terms of ongoing pregnancy (40%, P < 0.05) and implantation rates (18%, P < 0.05) in women with euploid embryos available for transfer with the first and second IVF/PGD cycles, compared to women with no euploid embryos available for transfer with either the first or second cycle. The positive predictive value of the first euploid cycle predicting a second euploid cycle was 72%, 95% CI 0.66–0.78. The negative predictive value of an aneuploid cycle was 50%, 95% CI 0.27–0.72. The sensitivity and specificity of the first PGD cycle predicting the second was 84%, 95% CI 0.77–0.91 and 33%, 95% CI 0.18–0.48, respectively.
Even with a history of recurrent implantation failure, the availability of euploid embryos, especially on two, consecutive PGD cycles is associated with high ongoing pregnancy and implantation rates. Conversely, the absence of euploid embryos for transfer predicts poor reproductive outcome, even if subsequent cycles do yield euploid embryos.
PMCID: PMC2582115  PMID: 18266102
Aneuploidy; Recurrent IVF failure; Multicolor FISH; Preimplantation genetic diagnosis
22.  Successful implantation and live birth of a healthy boy after triple biopsy and double vitrification of oocyte-embryo-blastocyst 
SpringerPlus  2015;4:22.
Preimplantation genetic diagnosis and/or screening (PGD/PGS) allow the assessment of the genetic health of an embryo before transferring it into the uterus. These techniques require the removal of cellular material (polar bodies, blastomere(s) or trophectoderm cells) in order to perform the proper genetic analysis. We report the implantation and live birth outcome of a vitrified-warmed blastocyst developed after triple biopsy and double vitrification procedures at oocyte, cleavage embryo and blastocyst stage.
Case description
An infertile couple, with family history of β-thalassemia, searched for IVF procedure and PGD. First polar bodies biopsy with subsequent vitrification was uninformative due to meiotic crossing-over, so oocytes were inseminated after warming. Two embryos were obtained and blastomere biopsy was performed on day 3 with inconclusive results on their genetic status. Their culture resulted in one expanded blastocyst stage on day 7 that underwent trophectoderm biopsy and vitrification. This embryo showed to be normal. It was then warmed and transferred in an artificial cycle.
Discussion and Evaluation
Preconception genetic analysis by removal and analysis of the first polar body is technically possible, but the genetic information that we can obtain at this stage may be limited and the oocytes to be inseminated is not predictable. Compared to blastomere biopsy, trophectoderm biopsy has more diagnostic efficiency with respect to both chromosomal mosaicism and PCR accuracy, reducing the problems of amplification failure and allele drop out. Moreover, embryos biopsied at the cleavage stage seem to have lower implantation rate than biopsied blastocyst.
This is the first case report of a live birth obtained from a three step biopsy and double vitrification procedures of a blastocyst. This case report seems also to suggest the harmlessness of all these procedures if carefully performed by a skilled biologist in an IVF lab with quality management system. Finally, our study highlight that blastocyst cryopreserved on day 7 have clinically important potential and embryos that not reach blastocyst stage on day 6 should not to be discharged because they may result in an ongoing pregnancy.
PMCID: PMC4300308  PMID: 25625041
Vitrification; Polar body biopsy; Embryo biopsy; Blastocyst biopsy
23.  Polar body based aneuploidy screening is poorly predictive of embryo ploidy and reproductive potential 
Polar body (polar body) biopsy represents one possible solution to performing comprehensive chromosome screening (CCS). This study adds to what is known about the predictive value of polar body based testing for the genetic status of the resulting embryo, but more importantly, provides the first evaluation of the predictive value for actual clinical outcomes after embryo transfer.
SNP array was performed on first polar body, second polar body, and either a blastomere or trophectoderm biopsy, or the entire arrested embryo. Concordance of the polar body-based prediction with the observed diagnoses in the embryos was assessed. In addition, the predictive value of the polar body -based diagnosis for the specific clinical outcome of transferred embryos was evaluated through the use of DNA fingerprinting to track individual embryos.
There were 459 embryos analyzed from 96 patients with a mean maternal age of 35.3. The polar body-based predictive value for the embryo based diagnosis was 70.3 %. The blastocyst implantation predictive value of a euploid trophectoderm was higher than from euploid polar bodies (51 % versus 40 %). The cleavage stage embryo implantation predictive value of a euploid blastomere was also higher than from euploid polar bodies (31 % versus 22 %).
Polar body based aneuploidy screening results were less predictive of actual clinical outcomes than direct embryo assessment and may not be adequate to improve sustained implantation rates. In nearly one-third of cases the polar body based analysis failed to predict the ploidy of the embryo. This imprecision may hinder efforts for polar body based CCS to improve IVF clinical outcomes.
PMCID: PMC4156943  PMID: 25106935
Preimplantation genetic screening (PGS); Polar bodies; Aneuploidy; Blastocyst; SNP array
24.  Polar body array CGH for prediction of the status of the corresponding oocyte. Part I: clinical results 
Human Reproduction (Oxford, England)  2011;26(11):3173-3180.
Several randomized controlled trials have not shown a benefit from preimplantation genetic screening (PGS) biopsy of cleavage-stage embryos and assessment of up to 10 chromosomes for aneuploidy. Therefore, a proof-of-principle study was planned to determine the reliability of alternative form of PGS, i.e. PGS by polar body (PB) biopsy, with whole genome amplification and microarray-based comparative genomic hybridization (array CGH) analysis.
In two centres, all mature metaphase II oocytes from patients who consented to the study were fertilized by ICSI. The first and second PBs (PB1and PB2) were biopsied and analysed separately for chromosome copy number by array CGH. If either or both of the PBs were found to be aneuploid, the corresponding zygote was then also processed by array CGH for concordance analysis.
Both PBs were biopsied from a total of 226 zygotes from 42 cycles (average 5.5 per cycle; range 1–15) in 41 couples with an average maternal age of 40.0 years. Of these, the ploidy status of the zygote could be predicted in 195 (86%): 55 were euploid (28%) and 140 were aneuploid (72%). With only one exception, there was at least one predicted aneuploid zygote in each cycle and in 19 out of 42 cycles (45%), all zygotes were predicted to be aneuploid. Fresh embryos were transferred in the remaining 23 cycles (55%), and one frozen transfer was done. Eight patients had a clinical pregnancy of which seven were evolutive (ongoing pregnancy rates: 17% per cycle and 30% per transfer). The ploidy status of 156 zygotes was successfully analysed by array CGH: 38 (24%) were euploid and 118 (76%) were aneuploid. In 138 cases complete information was available on both PBs and the corresponding zygotes. In 130 (94%), the ploidy status of the zygote was concordant with the ploidy status of the PBs and in 8 (6%), the results were discordant.
This proof-of-principle study indicates that the ploidy of the zygote can be predicted with acceptable accuracy by array CGH analysis of both PBs.
PMCID: PMC3196878  PMID: 21908463
microarray CGH analysis; polar body; oocyte; zygote; PGS
25.  Lessons from elective in vitro fertilization (IVF) in, principally, non-infertile women 
We here report the first investigation of exclusively elective in vitro fertilization (IVF) cycles in women with no apparent history of infertility. Since IVF outcome in women with infertility are always influenced by underlying causes of infertility, a study on non-infertile women may offer new insights.
We investigated 88 females without history of infertility in 109 consecutive elective IVF cycles, almost exclusively performed for purposes of preimplantation genetic screening (PGS; i.e., elective gender selection). The following questions were addressed: (i) impact of PGS on IVF pregnancy chances; (ii) impact of transfer of 1 vs. ≥2 embryos on IVF pregnancy chances; (iii) correlation of anti-Müllerian hormone (AMH) levels to embryo ploidy (iv) effect of gonadotropin dosage used in stimulation on available embryos for transfer; and (v) in form of a 1:1 case control study, compared 33 elective PGS cycles with matched control cycles without PGS, performed in couples with either prior tubal ligations and/or severe male factor infertility as indication of IVF.
The overall clinical pregnancy rate for the group was 36.7%; pregnancy was associated with number of euploid (P = 0.009) and number of embryos transferred (P = 0.001). Odds of pregnancy were 3.4-times higher if ≥4 euploid embryos were produced in comparison to <4 (95% CI 1.2 to 9.2; P = 0.019), and odds of pregnancy were 6.6-times higher if greater than or equal to 2 rather than <1 euploid embryos were transferred (95% CI 2.0 to 21.7; P = 0.002). Increasing AMH (P = 0.001) and gonadotropin dosage used in ovarian stimulation (P = 0.024), was, independently, associated with number of available euploid embryos. Increasing AMH, but not follicle stimulating hormone (FSH), was associated with number of embryos available for biopsy and PGS (P = 0.0001). Implantation rates were 26.4% with PGS and 9.5% without (P = 0.008). Women undergoing PGS, demonstrated 4.58-times higher odds of pregnancy than matched controls (95% CI 1.102 to 19.060, Exp 4.584, P = 0.036).
This study suggests that outcomes of elective IVF cycles may significantly deviate from infertility-associated cycles. Affirming proof of concept for PGS, utilizing day-3 embryo biopsy and fluorescence in-situ hybridization (FISH), both widely held responsible for earlier failures to establish such proof, suggests that the principal cause of prior failures were likely not insufficient laboratory techniques but poor patient selection for PGS. Such a conclusion questions the current reintroduction of PGS with improved techniques and technologies in absence of prior determination of suited patient populations.
PMCID: PMC3495227  PMID: 22716082
In vitro fertilization (IVF); Preimplantation genetic diagnosis (PGD); Preimplantation genetic screening (PGS); Aneuploidy; Pregnancy rates; Gonadotropin dosage; Anti-Müllerian hormone (AMH); Single embryo transfer

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