Preliminary assessment of aneuploidy rates between the polar, mid and mural trophectoderm.

The objective of this study is to compare aneuploidy rates between three distinct areas of the human trophectoderm: mural, polar and a region in between these two locations termed the 'mid' trophectoderm. This is a cohort study on in vitro fertilization (IVF) patients undergoing comprehensive chromosome screening at the blastocyst stage at a private IVF clinic. All embryos underwent assisted hatching on day 3 with blastocyst biopsy and comprehensive chromosome screening. Biopsied blastocysts were divided into three groups depending on which area (polar, mid, or mural) of the trophectoderm was protruding from the zona pellucida and biopsied. Aneuploidy rates were significantly higher with cells from the polar region of the trophectoderm (56.2%) compared with cells removed from the mural region of the trophectoderm (30.0%; P = 0.0243). A comparison of all three areas combined also showed a decreasing trend, but this did not reach clinical significance, polar (56.2%), mid (47.4%) and mural trophectoderm (30.0%; P = 0.1859). The non-concordance demonstrated between polar and mural trophectoderm can be attributed to biological occurrences including chromosomal mosaicism or procedural differences between embryologists.

Comparison of aneuploidy, pregnancy and live birth rates between day 5 and day 6 blastocysts.

Comprehensive chromosome screening is typically used for aneuploidy analysis of blastocysts. It is believed that either day of blastocyst development is acceptable. Euploidy rates and outcomes were examined between day 5 and day 6 blastocysts in two studies. First, euploidy rates of day 5 and day 6 blastocysts were examined on a per-embryo and per-patient basis. Second, outcomes were compared when only euploid day 5 or day 6 blastocysts were transferred in a cryopreserved embryo transfer cycle. In cycles (n = 70) that had blastocysts biopsied on both day 5 and day 6, day 5 blastocysts had a higher chance of being euploid than day 6 blastocysts (125/229 [54.6%]) and (77/180 [42.8%]), respectively (P = 0.0231). Similarly, euploid rates in blastocysts from patients (n = 193) with day 5 biopsy, day 6 biopsy, or both, were significantly higher in day 5 (235/421 [55.8%]) compared with day 6 (184/413 [44.6%]) blastocysts (P = 0.0014). In the second study, 50 women (36.1 ± 4.3 years) and 39 women (35.1 ± 3.8 years) with only euploid day 5 or euploid day 6 blastocysts transferred during a cryopreserved embryo transfer had similar cycle outcomes. Although underpowered, these data suggest that euploid day 6 blastocysts are as capable of positive outcomes as their euploid day 5 counterparts.

Outcomes of blastocysts biopsied and vitrified once versus those cryopreserved twice for euploid blastocyst transfer.

Trophectoderm biopsy with comprehensive chromosome screening (CCS) has been shown to increase implantation and pregnancy rates. Some patients desire CCS on previously cryopreserved blastocysts, resulting in blastocysts that are thawed/warmed, biopsied, vitrified and then warmed again. The effect of two cryopreservation procedures and two thawing/warming procedures on outcomes has not been effectively studied. Cycles were divided into two groups: group 1 patients underwent a cryopreserved embryo transfer with euploid blastocysts that were vitrified and warmed once; group 2 patients had a cryopreserved embryo transfer of a euploid blastocyst that was cryopreserved, thawed/warmed, biopsied, vitrified and warmed. Groups 1 and 2 included 85 and 17 women aged 35.6 ± 3.9 and 35.3 ± 4.9 years, respectively (not significantly different). Blastocyst survival in group 1 (114/116, 98.3%) and survival of second warming in group 2 (21/24, 87.5%) was significantly different (P = 0.0354). There was no difference between biochemical (68.2% and 62.5%) and clinical (61.2% and 56.3%) pregnancy rates, implantation rate (58.4% and 52.4%) and live birth/ongoing pregnancy rate (54.0% and 47.6%) between groups 1 and 2, respectively. Although it is unconventional to thaw/warm, biopsy, revitrify and rewarm blastocysts for cryopreserved embryo transfer, the results indicate that outcomes are not compromised. Trophectoderm biopsy and screening the embryos for chromosomal abnormalities has been reported to increase implantation and pregnancy rates. There is a category of patients requesting chromosomal screening on previously cryopreserved blastocysts. This scenario requires blastocysts to be thawed/warmed, biopsied, cryopreserved, and thawed/warmed again. The effect of double cryopreservation procedures and double thawing/warming procedures on pregnancy is unknown. Patients were divided into two groups, group 1 underwent a cryopreserved embryo transfer with a chromosomally normal blastocyst that was vitrified and warmed once and group 2 included patients that had a cryopreserved embryo transfer of a chromosomally normal blastocyst that was cryopreserved, thawed/warmed, biopsied, vitrified, and rewarmed. A total of 85 and 17 women aged 35.6 ± 3.9 and 35.3 ± 4.9 years were included in groups 1 and 2, respectively. The survival rate for group 1 (114 of 116, 98.3%) compared with the second warming for group 2 (21 of 24, 87.5%) was significantly higher. There was no difference between biochemical (68.2% and 62.5%), and clinical pregnancies (61.2% and 56.3%), implantation (58.4% and 52.4%), and live birth/ongoing rates (54.0% and 47.6%) between groups 1 and 2. Although it is unconventional to twice cryopreserve and twice thaw/warm a blastocyst, our results indicate that outcomes are not compromised.

Structural integrity and developmental potential of spermatozoa following microwave-assisted drying in the domestic cat model.

Characterizing the resilience of mammalian cells to non-physiological conditions is necessary to develop preservation and long-term storage strategies at low or ambient temperatures. Using the domestic cat model, the objective of the study was to characterize structural integrity (morphology and DNA damage) as well as functional properties (sperm aster formation and embryo formation after sperm injection) of spermatozoa after microwave-assisted drying to a moisture content compatible with storage in a glassy state at supra-zero temperatures. In Experiment 1, cat epididymal spermatozoa were porated with hemolysin and dried (using a commercial microwave oven set to 20% power) in the presence of trehalose for up to 50 min in a low humidity environment (11%) before measuring moisture content and sample temperature. In Experiment 2, morphology and DNA integrity were evaluated in sperm dried for up to 30 min (using the same method as above) versus fresh spermatozoa. In Experiment 3, the functionality of sperm dried for 30 min versus fresh sperm cells was evaluated after injection into oocytes based on sperm aster formation (5 h post-injection) and embryo development in vitro over 7 days. Moisture contents compatible with dry state storage were reached after 30 min of microwave-assisted drying. After rehydration, sperm morphology was not affected and the percentages of cells with damaged DNA (∼6.5%) was similar to the fresh controls. Sperm aster diameters appeared to be generally smaller for dried-rehydrated cells compared to the fresh controls. This observation was consistent with a lower proportion of blastocyst formation after injection with dried spermatozoa (6.5%) compared to fresh spermatozoa (15%). However, the blastocyst quality based on the total blastomere number was not affected by the sperm treatment. This is the first and encouraging report in any species so far demonstrating that spermatozoa can be dried using microwaves without causing irreversible damage to the cellular structure and function.

The origin, mechanisms, incidence and clinical consequences of chromosomal mosaicism in humans.

BACKGROUND: Chromosomal mosaicism, the presence of two or more distinct cell lines, is prevalent throughout human pre- and post-implantation development and can lead to genetic abnormalities, miscarriages, stillbirths or live births. Due to the prevalence and significance of mosaicism in the human species, it is important to understand the origins, mechanisms and incidence of mosaicism throughout development. METHODS: Literature searches were conducted utilizing Pubmed, with emphasis on human pre- and post-implantation mosaicism. RESULTS: Mosaicism persists in two separate forms: general and confined. General mosaicism is routine during human embryonic growth as detected by preimplantation genetic screening at either the cleavage or blastocyst stage, leading to mosaicism within both the placenta and fetus proper. Confined mosaicism has been reported in the brain, gonads and placenta, amongst other places. Mosaicism is derived from a variety of mechanisms including chromosome non-disjunction, anaphase lagging or endoreplication. Anaphase lagging has been implicated as the main process by which mosaicism arises in the preimplantation embryo. Furthermore, mosaicism can be caused by any one of numerous factors from paternal, maternal or exogenous factors such as culture media or possibly controlled ovarian hyperstimulation during in vitro fertilization (IVF). Mosaicism has been reported in as high as 70 and 90% of cleavage- and blastocyst-stage embryos derived from IVF, respectively. CONCLUSIONS: The clinical consequences of mosaicism depend on which chromosome is involved, and when and where an error occurs. Mitotic rescue of a meiotic error or a very early mitotic error will typically lead to general mosaicism while a mitotic error at a specific cell lineage point typically leads to confined mosaicism. The clinical consequences of mosaicism are dependent on numerous aspects, with the consequences being unique for each event.

Blastocyst euploidy and implantation rates in a young (<35 years) and old (≥35 years) presumed fertile and infertile patient population.

OBJECTIVE: To examine the relationship between blastocyst euploidy and implantation rates in a presumed fertile patient population. DESIGN: Retrospective analysis. SETTING: Private IVF clinic. PATIENT(S): IVF patients undergoing comprehensive chromosome screening (CCS). INTERVENTION(S): Embryo biopsy at the blastocyst stage with preimplantation genetic screening using CCS. MAIN OUTCOME MEASURE(S): Euploidy, chemical pregnancy, and implantation rates. RESULT(S): There was no significant difference in the number of euploid blastocysts between presumed fertile (68/118, 57.6%) and infertile (75/132, 56.8%) patients<35 years old. Likewise, there was no significant difference in the number of euploid blastocysts between presumed fertile (42/86, 48.8%) and infertile (97/206, 47.1%) patients≥35 years old. When those same patients underwent a corresponding frozen embryo transfer cycle, presumed fertile patients demonstrated a significantly higher chemical pregnancy rate when compared with infertile patients, 28/33 (84.8%) and 50/81 (61.7%), respectively. Moreover, presumed fertile patients exhibited significantly higher implantation rates compared with infertile patients, 36/42 (85.7%) and 54/109 (66.7%), respectively. CONCLUSION(S): When subdivided by maternal age, no significant difference was seen in blastocyst euploidy rates between presumed fertile and infertile patients; however, chemical pregnancy and implantation rates were significantly higher in a presumed fertile patient population even when transferring only euploid blastocysts. This would indicate that infertility, as a disease, may encompass other aspects such as uterine or other unknown embryological factors that can influence outcomes.