Kangpu Xu

Embryo development after heterotopic transplantation of cryopreserved ovarian tissue

BACKGROUND Cancer treatments, including chemotherapy, radiotherapy, and radical surgery, can induce premature menopause and infertility in hundreds of thousands of women of reproductive age every year. One of the ways to possibly preserve fertility before these treatments is to cryopreserve ovarian tissue for later transplantation. We aimed to restore fertility by cryopreservation and transplantation of ovarian tissue. METHODS Ovarian tissue was cryopreserved from a 30-year-old woman with breast cancer before chemotherapy-induced menopause, and this tissue was transplanted beneath the skin of her abdomen 6 years later. FINDINGS Ovarian function returned in the patient 3 months after transplantation, as shown by follicle development and oestrogen production. The patient underwent eight oocyte retrievals percutaneously and 20 oocytes were retrieved. Of the eight oocytes suitable for in-vitro fertilisation, one fertilised normally and developed into a four-cell embryo. INTERPRETATION Fertility and ovarian endocrine function can be preserved in women by long-term ovarian tissue banking.

Preimplantation genetic diagnosis for retinoblastoma: the first reported liveborn.

PURPOSE To develop an accurate mutation analysis procedure for retinoblastoma gene (RB1) mutation, which is sensitive at the single-cell level, and to use in vitro fertilization (IVF) and preimplantation genetic diagnosis (PGD) to achieve pregnancies without retinoblastoma. DESIGN Case report. METHODS Twelve day 3 embryos, obtained by IVF with intracytoplasmic sperm injection, underwent single-cell DNA testing via polymerase chain reaction and restriction enzyme analysis to detect the presence of a paternal RB1 mutation. Embryos were diagnosed as being unaffected and were transferred to the uterus on day 5. MAIN OUTCOME MEASURES Achieving a healthy pregnancy and delivery, assessed by clinical presentation, fundus photography, and RB1 molecular analysis. RESULTS A singleton pregnancy was achieved, and a child without retinoblastoma was born. The absence of the paternal RB1 mutation was confirmed on a sample of peripheral blood from the newborn. CONCLUSIONS We are first to report a successful human liveborn, delivered after IVF with preimplantation genetic diagnosis for retinoblastoma. The successful result indicates that preimplantation genetic diagnosis exists for this genetic disease and may represent a viable alternative to prenatal diagnosis with the subsequent option of terminating an affected pregnancy.

Evaluation of genome coverage and fidelity of multiple displacement amplification from single cells by SNP array.

The scarce amount of DNA contained in a singe cell is a limiting factor for clinical application of preimplantation genetic diagnosis mainly due to the risk of misdiagnosis caused by allele dropout and the difficulty in obtaining copy number variations in all 23 pairs of chromosomes. Multiple displacement amplification (MDA) has been reported to generate large quantity of products from small amount of templates. Here, we evaluated the fidelity of whole-genome amplification MDA from single or a few cells and determined the accuracy of chromosome copy number assessment on these MDA products using an Affymetrix 10K 2.0 SNP Mapping Array. An average coverage rate (86.2%) from single cells was obtained and the rates increased significantly when five or more cells were used as templates. Higher concordance for chromosome copy number from single cells could be achieved when the MDA amplified product was used as reference (93.1%) than when gDNA used as reference (82.8%). The present study indicates that satisfactory genome coverage can be obtained from single-cell MDA which may be used for studies where only a minute amount of genetic materials is available. Clinically, MDA coupled with SNP mapping array may provide a reliable and accurate method for chromosome copy number analysis and most likely for the detection of single-gene disorders as well.

Morphologic grading of euploid blastocysts influences implantation and ongoing pregnancy rates

OBJECTIVE To determine whether blastocyst grading can predict pregnancy outcomes in the frozen-thawed embryo transfer (FET) of euploid blastocysts. DESIGN Retrospective cohort study. SETTING Academic medical center. PATIENT(S) Women who underwent FET of euploid embryo(s) between January 2013 and December 2015, with blastocysts were divided into four groups based on their morphologic grading before cryopreservation: excellent (≥3AA), good (3-6AB, 3-6BA, 1-2AA), average (3-6BB, 3-6AC, 3-6CA, 1-2AB, 1-2BA), and poor (1-6BC, 1-6CB, 1-6CC, 1-2BB). INTERVENTION(S) FET. MAIN OUTCOMES MEASURE(S) Ongoing pregnancy rate (OPR). RESULT(S) A total of 417 FET cycles (477 embryos) were included. Excellent-quality embryos (n = 38) yielded a statistically significantly higher OPR than poor-quality embryos (n = 106) (84.2% vs. 35.8%; adjusted odds ratio 11.0; 95% confidence interval, 3.8-32.1) and average-quality embryos (n = 197) (84.2% vs. 55.8%; adjusted odds ratio 4.8; 95% confidence interval, 1.7-13.3). Good-quality embryos (n = 76) were associated with a statistically significantly higher OPR than poor-quality embryos (61.8% vs. 35.8%). These odds ratios were adjusted for patients age, body mass index, number of transferred embryos, type of frozen cycle, peak endometrial thickness, day of trophectoderm biopsy (5 or 6), and total number of euploid embryos for each patient. An inner cell mass grade of A yielded a statistically significantly higher OPR than ICM grade C (76.2% vs. 13.5%) or grade B (76.2% vs. 53.6%) after controlling for all confounders. CONCLUSION(S) Contrary to prior published studies, the current data suggest that blastocyst morphologic grading and particularly inner cell mass grade is a useful predictor of OPR per euploid embryo. Morphologic grading should be used to help in the selection among euploid blastocysts.

Improving the Fixation Method for Preimplantation Genetic Diagnosis by Fluorescent in Situ Hybridization

Purpose:Our purpose was to modify a fixation method using Tween-20 and HCl (TH) and to compare it with a protocol using methanol and acetic acid (MA) for the improvement of preimplantation genetic diagnosis by fluorescence in situ hybridization (FISH).Methods:Single blastomeres were allocated to either the TH or the MA procedure. The two methods were compared to evaluate efficiency of fixation and the intensities of FISH signals.Results:With the TH method, 123 (93.9%) of 131 blastomeres were fixed, while only 95 (78.5%) of 121 were fixed with MA. Average scores for the intensity of FISH signals were significantly stronger for TH than for MA (P < 0.05). There was also a significant difference in signal intensity scores between the two methods for type-3 nuclei.Conclusions:Our results indicate that not only are fewer nuclei lost during fixation but also stronger FISH signals can be obtained with the TH method. Thus, modified TH can improve the overall efficiency of preimplantation genetic diagnosis.

The importance of cytoplasm in early embryonic development

AbstractPurpose: A mouse model was used to evaluate the consequences of partial cytoplasm removal, which mimics the consequence of human early embryo fragmentation, at the one-cell stage on embryonic development to the morula and blastocyst stages. Results: Opening the zona pellucida at the zygote stage has no detrimental effect on the number of differentiated or total cells at morulalblastocyst stage. However, reduced cytoplasm significantly affects the number of inner cell mass cells, 21.7 (10% cytoplasm removed) vs 27.6 (control) and 15.1 (20% cytoplasm removed) vs 24.7 (control). Conclusions: The contribution of cytoplasm of maternal origin is critical to early embryonic development.

CGG repeat length and AGG interruptions as indicators of fragile X–associated diminished ovarian reserve

PurposeFragile X premutation (PM) carriers may experience difficulties conceiving a child probably due to fragile X–associated diminished ovarian reserve (FXDOR). We investigated which subgroups of carriers with a PM are at higher risk of FXDOR, and whether the number of AGG interruptions within the repeat sequence further ameliorates the risk.MethodsWe compared markers of ovarian reserve, including anti-Müllerian hormone, antral follicle count, and number of oocytes retrieved between different subgroups of patients with a PM.ResultsWe found that carriers with midrange repeats size (70–90 CGG) demonstrate significantly lower ovarian reserve. Additionally, the number of AGG interruptions directly correlated with parameters of ovarian reserve. Patients with longer uninterrupted CGG repeats post–AGG interruptions had the lowest ovarian reserve.ConclusionThis study connects AGG interruptions and certain CGG repeat length to reduced ovarian reserve in carriers with a PM. A possible explanation for our findings is the proposed gonadotoxicity of the FMR1 transcripts. Reduction of AGG interruptions could increase the likelihood that secondary RNA structures in the FMR1 messenger RNA are formed, which could cause cell dysfunction within the ovaries. These findings may provide women with guidance regarding their fertility potential and accordingly assist with their family planning.

Single-nucleotide polymorphism array coupled with multiple displacement amplification: accuracy and spatial resolution for analysis of chromosome copy numbers in few cells.

When coupled with multiple displacement amplification (MDA), microarray‐based comparative genomic intensity allows detection of chromosome copy number aberrations even in single or few cells, but the actual performance of the system and their influencing factors have not been well defined. Here, using single‐nucleotide polymorphism (SNP) array, we analyzed copy number profiles from DNA amplified by MDA in 1–10 cells and estimated the accuracy and spatial resolution of the analysis. Based on the concordance of SNP copy numbers for DNA with and without MDA, the accuracy of the system can be significantly enhanced by using MDA‐amplified DNA as reference and also by increasing the cell numbers. Analyses under different smoothing treatments revealed a practical resolution of 2 Mb for 10 cells and 10 Mb for a single cell. When both cells with known chromosomal duplication and deletion were analyzed, this platform detected a copy number “loss” more accurately than a “gain” (P < 0.01), particularly in single‐cell MDA products. Together, we demonstrated that SNP array coupled with MDA is reliable and efficient for detection of copy number aberrations in a small number of cells, and its accuracy and resolution can both be significantly enhanced with increasing the number of cells as MDA template.

Blastocyst development rate influences implantation and live birth rates of similarly graded euploid blastocysts

OBJECTIVE To determine whether the blastocyst development rate, as assessed by the day of trophectoderm biopsy (day 5 vs. day 6), affects the live birth rate (LBR) of similarly graded euploid blastocysts. DESIGN Retrospective cohort study. SETTING Academic medical center. PATIENT(S) Patients who underwent frozen-thawed single euploid blastocyst transfers from 2013 to 2016 were included. Blastocyst morphologic grading was performed on day 5 or day 6 before the biopsy, with embryos designated into the following groups: good (3-6AA, 3-6AB, and 3-6BA), average (2-6BB), and poor (2-6BC and 2-6CB). INTERVENTION(S) Frozen-thawed embryo transfer. MAIN OUTCOME MEASURE(S) Implantation rate (IR) and LBR. RESULT(S) A total of 701 frozen-thawed single euploid blastocyst transfer cycles were included. Cycles in which day 5 blastocysts were transferred (n = 366) were associated with a significantly higher LBR than those in which day 6 blastocysts were transferred (n = 335; 60.4% vs. 44.8%). The odds ratio remained significant after controlling for all confounders, including the blastocyst grading. Furthermore, there was a significant difference in LBRs between good-quality, average-quality, and poor-quality blastocysts (67.8%, 53.4%, and 29.5%, respectively). Embryos reaching good-quality blastocysts on day 5 yielded significantly higher LBR (72.8% vs. 56.5%) and IR (77.7% vs. 58.7%) compared with those reaching similar quality blastocysts on day 6. Similarly, day 5 average-quality embryos conveyed a significantly higher IR compared with day 6 embryos of the same quality (64.4% vs. 53.4%). CONCLUSION(S) In addition to aneuploidy assessment, the speed of embryo development to the blastocyst stage and an evaluation of blastocyst morphology are critical to selecting the best embryo.