Embryo Fragmentation Research Articles

Physical properties of terrestrial planets and water delivery in the habitable zone using N-body simulations with fragmentation

Aims. The goal of this research is to study how the fragmentation of planetary embryos can affect the physical and dynamical properties of terrestrial planets around solar-type stars. Our study focuses on the formation and evolution of planets and water delivery in the habitable zone (HZ). We distinguish class A and class B HZ planets, which have an accretion seed initially located inside and beyond the snow line, respectively. Methods. We developed an N-body integrator that incorporates fragmentation and hit-and-run collisions, which is called D3 N-body code. From this, we performed 46 numerical simulations of planetary accretion in systems that host two gaseous giants similar to Jupiter and Saturn. We compared two sets of 23 N-body simulations, one of which includes a realistic collisional treatment and the other one models all impacts as perfect mergers. Results. The final masses of the HZ planets formed in runs with fragmentation are about 15–20% lower than those obtained without fragmentation. As for the class A HZ planets, those formed in simulations without fragmentation experience very significant increases in mass with respect to their initial values, while the growth of those produced in runs with fragmentation is less relevant. We remark that the fragments play a secondary role in the masses of the class A HZ planets, providing less than 30% of their final values. In runs without fragmentation, the final fraction of water of the class A HZ planets keeps the initial value since they do not accrete water-rich embryos. In runs with fragmentation, the final fraction of water of such planets strongly depends on the model used to distribute the water after each collision. The class B HZ planets do not show significant differences concerning their final water contents in runs with and without fragmentation. From this, we find that the collisional fragmentation is not a barrier to the survival of water worlds in the HZ.

Embryo fragmentation and its relationship with aneuploidy

Objective: Selecting embryos is an important step in the in vitro fertilization process before transferring them to the uterus. There are some invasive methods for choosing a good quality embryo, such as embryo grading. This method evaluates the equality and fragmentation of an embryo. However, this method does not adequately evaluate the chromosomal status of the embryos, which is often necessary for high-risk embryos. Here, we evaluated embryo fragmentation and chromosomal numbers using next-generation sequencing. Materials and Methods: Each embryo was biopsied on the 3rd or 5th culture day to obtain a single blastomere cell. DNA was then extracted from each blastomere and whole-genome amplification was carried out. Amplification products were then sequenced to obtain a ploidy number. Results: Among the 30 embryos that were evaluated, 19 embryos had no fragments, 10 embryos had small fragments, and 1 embryo had moderate fragments. However, 12 of 19 embryos, 57.9% with no fragments were detected to have chromosomal abnormality. Aneuploidy was increased in 7 of 10 embryos (70%) with mild fragments. One moderately fragmented embryo included was surprisingly found to have normal ploidy (100%). Gamma correlation test showed that there was no correlation between fragmentation and the incidence rate of aneuploidy (P > 0.05). Although there was no correlation, the study's result exemplifies that aneuploidy rate increased along with higher fragmentation. Conclusion: This research concluded that embryo fragmentation was not correlated with aneuploidy.

Cooling of in vitro-produced bovine embryos: Effects of medium and time on gene expression, DNA fragmentation and embryonic survival

The aim of this study was to investigate the effects of Medium 199 (M199) and BotuEmbryo® (BE) as well as the time (24 an 48 h) of cooling at 5 °C on the transcript level of genes related to bovine embryonic metabolism (GLUT1) and cellular stress (HSPA1A, PRDX1 and SOD1), on the rate of blastocyst re-expansion and hatch, embryo survival, total number of cells and DNA fragmentation in IVP bovine embryos. There was an overall reduction in embryo survival and development to hatching/hatched stage with the increase in cooling time, especially in those cooled in M199. Although TUNEL-positive cells were identified in both cooled and NC embryos, a higher number of apoptotic cells was observed in BE-48 than in NC and BE-24. However, no effect of either medium or cooling time was found among all groups for the set of gene transcripts. In conclusion, no detrimental effect on the expression of genes as well as embryonic cell proliferation was remarkable in cooling of IVP bovine embryos. However, according to the in vitro predictive assessment of embryo viability, the cooling of IVP bovine embryos in the evaluated media appears to be feasible for 24 h but not for 48 h.

The impact of in vitro human embryo fragmentation on blastocyst development and ploidy using Next-Generation Sequencing (NGS)

Introduction The degree of fragmentation has been utilized in non-invasive selection of Day 2 or Day 3 embryos for transfer. Fragmentation happens in both in vitro and in vivo embryo, where anucleate cells of varying size and number are found along with nucleate cells in the embryo. While fragmentation may potentially affect the developmental potential of an embryo, very little evidence has been reported in the literature. This study was carried out to evaluate the effects of embryo fragmentation on the blastulation rate (BR), blastocyst utilization rate (BUR), implantation rate (IR) and euploid rate of the blastocyst. Materials & Methods A total of 438 PGS cycles from January 2017 to December 2017 were retrospectively analyzed. Intracytoplasmic sperm injection (ICSI) was carried out after oocyte retrieval. Normally fertilized (2PN) embryos (n= 3720) were evaluated according to the degree of fragmentation (group 0F: 0 – 5% fragments, group 0/1F: 6 -10% fragments, group 1F: 11 – 15% fragments, group 1/2F: 16 – 20% fragments, group 2F: 21 – 25% fragments and group 2/3F: >25% fragments) at 66 – 68 hours post injection. Following extended culture to blastocyst stage, trophectoderm (TE) biopsy was performed on good or fair graded blastocysts (n=1865) on either Day 5 or Day 6. Pre-implantation Genetic Screening (PGS) using Next-Generation Sequencing (NGS) (Veriseq Protocol, Illumina) was used for aneuploidy screening. A total of 252 euploid or low-level mosaicism blastocysts were transferred and the BR, BUR, IR and the euploidy rate of the blastocyst were evaluated. Results Embryos with higher degree of fragmentation have been found to have a lower BR (81.4%, 73.5%, 60.7%, 47.0%, 31.6% and 12.8%, respectively) and BUR (66.2%, 52.6%, 37.7%, 23.5%, 9.9% and 4.3%, respectively) in our study. We found that embryos with no fragment or lower degree of fragmentation have a higher chance of reaching the blastocyst stage, while most of the embryos with a higher degree of fragmentation were arrested after Day 3. Our study did not show any differences in euploidy rate, mosaicism rate and aneuploidy rate between groups with an average of 67.29%, 8.89% and 25.09% respectively. Transfer of blastocyst with higher degree of fragmentation on Day 3 did not show any significant decrease of in implantation rate. Conclusions In our study, fragmentation has been shown to affect blastocyst development due to unequal allocation of cells. But once a blastocyst develops, there do not seem to be any impact on IR and chromosome abnormalities. Indeed, pregnancies has been reported with the transfer of embryos with a high degree of fragmentation. Our small sample size however, may impact on the validity of this study. As embryo selection solely based on the degree of fragmentation on Day 3 is ineffective, extended culture of all embryos regardless of degree of fragmentation to blastocyst stage is recommended. This will potentially avoid accidental discarding of embryos that are destined to implant and form a pregnancy.

Antioxidants and glycine can improve the developmental competence of vitrified/warmed ovine immature oocytes.

Despite the numerous potential applications of oocyte cryopreservation, the poor success rate has limited its practical applications. In livestock, particularly in ovine, the oocytes have low developmental competence following vitrification/warming process. Considering the occurrence of osmotic and oxidative stresses during the vitrification/warming process, the application of antioxidants and osmolytes may improve the developmental competence of vitrified/warmed oocytes. In the present study, we aimed to evaluate the effects of the addition of ascorbic acid (AA) and N-acetyl cysteine (NAC) as antioxidants and glycine as an organic osmolyte either to the vitrification/warming solutions (VWS) or to the IVM medium on the developmental competence of vitrified/warmed ovine germinal vesicle stage oocytes. The survival rate in the vitrified groups was significantly lower than fresh ones. In vitrified/warmed oocytes, there was no significant difference in survival rate between supplemented and non-supplemented groups. The addition of AA and/or NAC to the VWS or IVM medium and adding glycine to the IVM medium reduced the proportion of apoptotic oocytes and fragmented embryos, which was reflected as an increase in the proportions of metaphase II stage oocytes and blastocyst production. The best result was achieved by supplementing the IVM medium with NAC. In our study condition, antioxidants and glycine could improve the developmental competence of vitrified/warmed ovine immature oocytes, especially when added during IVM.

Human zygote reconstruction by spindle, polar body or pronuclear transfer to treat repeated embryo fragmentation or embryo developmental arrest: The future is now

A common and very difficult issue to overcome as a clinical embryologist during an IVF cycle is the event of severe embryonic fragmentation or, more rarely, full developmental arrest. The current approach to bypass these phenomena is oocyte donation, but this raises several ethical concerns from the couple’s side, since the offspring will bear only 50% of the biological characteristics of the parents. Apart from spindle and polar body transfer, the emerging technique in order to overcome these obstacles is called Pronuclear Transfer (PNT). PNT is safest to perform with improved results than spindle or polar body transfer. In PNT, the two parental pronuclei are being transferred into a donor’s enucleated zygote and after a fusion process has been achieved, the newly constructed embryo may result into a newborn bearing both the paternal and maternal biological characteristics. This process opens a whole new horizon in assisted reproduction and will probably replace the traditional oocyte donation in the form we know it today.

41 Delineating the molecular connections between mitotic aneuploidy, micronucleation, and cellular fragmentation in pre-implantation bovine embryos

Whole chromosomal abnormalities (aneuploidy) that arise during early embryo development are a major contributor to in vitro fertilization failure. It is estimated that ~50 to 80% of human embryos contain aneuploid cells, which contribute to high levels of chromosomal mosaicism detected by pre-implantation genetic screening. Previous studies estimate that 32 to 88% of bovine embryos are aneuploid at the 2-cell stage, advocating cattle as a physiologically relevant model to study the mechanisms mediating meiotic and/or mitotic errors. In cleavage-stage human embryos, a process called cellular fragmentation is associated with aneuploidy, and when used in conjunction with assessment of early mitotic timing, can largely distinguish chromosomally normal and abnormal embryos. We recently demonstrated that some cellular fragments contain chromosomal material that likely began as mis-segregated chromosomes that were encapsulated into micronuclei. Given that bovine embryos exhibit cellular fragmentation, albeit to a lesser extent than human embryos, we hypothesise that cellular fragmentation is a response to micronucleation and represents a conserved mechanism to eliminate mis-segregated chromosomes from the pre-implantation embryo. Using a combination of live-cell imaging, single-cell DNA-sequencing, whole-embryo RNA-sequencing, quantitative RT-PCR, and multicolour confocal microscopy, we aim to further investigate the correlation between these phenomena using in vitro-produced bovine embryos. Similar to humans, the first three mitotic divisions are able to successfully predict progression to the blastocyst stage (N=84). Bovine embryos frequently contained multi-/micro-nuclei, and DNA-sequencing of individual bovine blastomeres up to 12 cells confirmed that ~58 to 87% of cleavage-stage bovine embryos are aneuploidy (N=38) and often detectable by abnormal cell divisions. Transcriptional profiling of fragmented versus non-fragmented bovine embryos via RNA-sequencing identified a small subset of differentially abundant genes at the 4-cell stage. Pathway analysis showed reduced abundance of genes associated with the cytoskeleton, microtubules, and spindle in 4-cell embryos with cellular fragmentation as well as enrichment of membrane targeting and vesicle fusion pathways. The potential role of these cellular components in micronucleation and cellular fragmentation is being assessed by microinjecting bovine zygotes with fluorescently labelled mRNA mCherry-H2B (chromatin marker) and mCitrine-LaminB1 (nuclear envelope marker), followed by overnight live-cell multicolour confocal imaging (Zeiss LSM 880 with AiryScan; Zeiss, Thornwood, NY, USA). Results from these studies contribute to our knowledge of early embryogenesis with translational application to help ameliorate embryonic loss in women and cattle.

Early fragment removal on in vitro fertilization day 2 significantly improves the subsequent development and clinical outcomes of fragmented human embryos.

ObjectiveTo determine whether fragment removal on in vitro fertilization (IVF) day 2 improved the subsequent development and pregnancy outcomes of fragmented embryos compared to similar-grade embryos without fragment removal.MethodsThis study was a retrospective analysis involving 191 IVF cycles in which all embryos had over 10% fragmentation (grade 3 or 4) on day 2 of the IVF-embryo transfer cycle from March 2015 to December 2017. IVF cycles were divided into the fragment removal group (n=87) and the no fragment removal group (n=104) as a control cohort. Before fragment removal, embryos with fragmentation on day 2 were incubated in Ca2+- and Mg2+-free biopsy medium under paraffin oil for 30 minutes. Microsurgical fragment removal was performed with later-assisted hatching and a handmade suction micropipette that had an outer diameter of 30 µm.ResultsThere were no significant differences in the characteristics of the patients between the control and the fragment removal groups. After fragment removal and subsequent in vitro culture for 24 hours, the number of blastomeres (7.1±1.7 vs. 6.9±1.6) was comparable between the transferred embryos in the two groups, but the morphological grade of the embryos in the fragment removal group (1.9±0.7) was significantly higher than that of the control group (3.1±0.5, p<0.01). The clinical pregnancy (43.7%) and implantation rates (25.8%) in the fragment removal group were significantly higher than those in the control group (28.8% and 14.0%, respectively; p<0.05).ConclusionEarly fragment removal on day 2 significantly improved the subsequent development and pregnancy outcomes of fragmented embryos.

Late embryo cleavage as an indicator of chromosome aneuploidy by pre-implantation genetic screening

During in vitro fertilization (IVF), not all patients receive good quality oocytes or embryos. This study investigated the frequency of aneuploidy and mosaicism in embryos obtained from parthenogenetic oocytes and poor morphological embryos that also exhibited late cleavage development by preimplantation genetic screening (PGS). Twenty late cleavage embryos under IVF treatment were cultured until day 5, and one of the blastomeres was biopsied. The embryos were divided into three groups (parthenogenetic oocytes and poor-quality embryos with dark cytoplasm, several vacuoles or fragments in the cytoplasm; arrested embryos; and good-quality embryos). All samples were tested for abnormalities using array-comparative genomic hybridization (a-CGH). Twenty late cleavage-stage embryos were biopsied and analyzed for aneuploidy. A total 17 of 20 (85%) embryos were aneuploid and three (15%) were euploid. The incidence of aneuploidy in good-quality embryos was 75%, while poor-quality embryos displayed a higher frequency (86.7%) of aneuploidy. The parthenogenetic oocytes were invariably aneuploid (100%). Based on the embryo-grading system by Veeck, embryo grades 1 and 2 exhibited a lower incidence of aneuploidy (75%), while grades 3 and 4 showed 1.33-times higher aneuploidy rates (RR = 1.33; p < 0.001). The numerical defects of the chromosomes increased with the percent of fragmentation, with fragmented embryos showing 1.32-times greater aneuploidy rates than the non-fragmented ones. Embryos with uneven blastomeres showed 1.07-times higher aneuploidy rate when compared with embryos showing even blastomeres (p < 0.0064). Late embryo cleavage results in a higher incidence of aneuploidy and mosaicism upon comparison with early embryo cleavage.

The impact of high progesterone levels on the day of HCG administration in assisted human reproduction treatments.

ObjectiveProgesterone is a steroid hormone that acts on the endometrium. It is known for producing physical and mood-related side effects. Few studies have looked into how progesterone levels affect embryo development and quality. This study aimed to find a cutoff level for serum progesterone on the day of HCG administration from which embryo quality is impaired.MethodsThe study included 145 cycles, from which 885 oocytes and 613 embryos were obtained. All patients had their serum progesterone levels measured on the day of HCG administration. Data sets were collected from patient medical records. The chi-square test was used to assess qualitative variables and the Mann-Whitney test to evaluate quantitative variables.ResultsStatistical analysis revealed that serum progesterone levels and reproductive variables were not significantly associated. In regards to oocyte maturity, however, when progesterone levels were greater than 1.3 ng/mL the probability of oocytes being immature increased by 12.7%. The fragmentation rate of embryos categorized as "top quality" in D3 increased proportionately to increases in progesterone levels (12.23%).ConclusionHigh progesterone levels appeared to be correlated with increased embryo fragmentation rates, but high serum levels of the hormone on the day of HCG administration had no impact on reproductive variables and were not associated with impaired embryo development.

New insights of subfertility among transplanted women: Immunosuppressive drug FK506 leads to calcium leak and oocyte activation before fertilization.

FKBP12, known as FK506 binding protein, binds to immunosuppressive drug FK506, which must be taken by patients who received organ transplant. The side effect of FK506 is that women have difficulties in bearing a baby, so it is important to find the reason of their subfertility. This research explored the expression of FKBP12 in pre-implantation embryos and investigated its potential effect on reproduction. The results demonstrate that FKBP12 had good co-localization with endoplasmic reticulum and inositol-1, 4, 5- trisphosphate receptor in pre-implantation stages. Inhibiting FKBP12 by FK506 significantly increased the rate of 1-cell and fragmented embryos, greatly reduced the rate of 2-cell embryos during in vitro fertilization. When the mice received FK506 by gavage for 21 days, the calcium intensity of oocytes was decreased, these mice were subfertile and gave birth to significantly less pups during 6-month breeding period. QPCR demonstrated that Fbxo43 and P27kip, which are related to the release of MII oocyte arrest, and calcium channel partner protein Orai1 were downregulated, while Cdc2 and Ca2+ sensor at ER, stromal interaction molecule 1 (Stim1) were upregulated for a short time after adding FK506. Further exploration discovered that FK506 treatment increased Ca2+ concentration in the cytoplasm and caused pronucleus formation, however, the rate of parthenogenetic activation was lower compared to SrCl2 group. These findings identify the previously unknown role of FKBP12 in female reproduction which contributes to the release of calcium via IP3 R channel and might open up new strategies for women who want to bear a baby after transplantation.

Efecto del uso de medio secuencial humano en la producción de blastocistos de hembra ovis canadensis mexicana por clonación manual

RESUMEN. Efecto del uso de medio secuencial humano en la producción de blastocistos de hembra Ovis canadensis mexicana por clonación manual interespecies. Desde el evidente problema del peligro de extinción que enfrenta un número significativo de especies silvestres a nivel mundial, se han aplicado biotecnologías encaminadas a promover su conservación y reproducción. Tal es el caso de la transferencia nuclear de células somáticas (TNCS) interespecies que ha sido aplicada y ha producido exitosamente embriones y crías nacidas vivas en algunas de estas especies. Sin embargo, la TNCS interespecies aún debe lidiar con otros problemas como la baja tasa de producción de embriones y de supervivencia de las crías nacidas (3-6%). Problemas que, entre otros, involucran la alta incidencia de fragmentación embrionaria. Los estudios son ahora dirigidos a resolver estos obstáculos, determinando el efecto de diferentes medios o suplementos utilizados en la producción in vitro de embriones. El objetivo de este estudio fue aplicar una variante de la TNCS interespecies (Clonación Manual-HMC) para la producción de embriones, determinando el efecto del uso de medios secuenciales humanos, en el borrego salvaje mexicano amenazado de extinción O. c. mexicana. Los resultados muestran que la producción de embriones incrementa con el uso de medios secuenciales humanos, permitiendo al embrión desarrollarse hasta la etapa de blastocisto (16% vs. 0%, P &lt; 0.05). Este es el primer reporte de clonación exitosa de embriones de hembra O. c. mexicana en México.

Developmental toxicity and DNA damaging properties of silver nanoparticles in the catfish (Clarias gariepinus)

Although, silver nanoparticles (AgNPs) are used in many different products, little information is known about their toxicity in tropical fish embryos. Therefore, this study evaluated the developmental toxicity of waterborne silver nanoparticles in embryos of Clarias gariepinus. Embryos were treated with (0, 25, 50, 75ng/L silver nanoparticles) in water up to 144h postfertilization stage (PFS). Results revealed various morphological malformations including notochord curvature and edema. The mortality rate, malformations, and DNA fragmentation in embryos exposed to silver nanoparticles increased in a dose- and embryonic stage-dependent manner. The total antioxidant capacity and the activity of catalase in embryos exposed to 25ng/L silver nanoparticles were decreased significantly while the total antioxidant capacity and the activity of catalase were insignificantly increased with increasing concentrations in the embryos from 24 to 144 h-PFS exposed to 50 and 75ng/L silver nanoparticles. Lipid peroxidation values showed fluctuations with doses of silver nanoparticles. Histopathological lesions including severely distorted and wrinkled notochord were observed. The current data propose that the toxicity of silver nanoparticles in C. gariepinus embryos is caused by oxidative stress and genotoxicity.

Alpha-linolenic acid protects the developmental capacity of bovine cumulus-oocyte complexes matured under lipotoxic conditions in vitro.

Elevated concentrations of free fatty acids (FFAs), predominantly palmitic, stearic, and oleic acids (PSO), exert detrimental effects on oocyte developmental competence. This study examined the effects of omega-3 alpha-linolenic acid (ALA) during in vitro oocyte maturation (IVM) in the presence of PSO on subsequent embryo development and quality, and the cellular mechanisms that might be involved. Bovine cumulus-oocyte complexes (COCs) were supplemented during IVM with ALA (50 μM), PSO (425 μM), or PSO+ALA. Compared with FFA-free controls (P<0.05), PSO increased embryo fragmentation and decreased good quality embryos on day 2 postfertilization. Day 7 blastocyst rate was also reduced. Day 8 blastocysts had lower cell counts and higher apoptosis but normal metabolic profile. In the PSO group, cumulus cell (CC) expansion was inhibited with an increased CC apoptosis while COC metabolism was not affected. Mitochondrial inner membrane potential (MMP; JC-1 staining) was reduced in the CCs and oocytes. Heat shock protein 70 (HSP70) but not glucose-regulated protein 78 kDa (GRP78, known as BiP; an endoplasmic reticulum stress marker) was upregulated in the CCs. Higher reactive oxygen species levels (DCHFDA staining) were detected in the oocytes. In contrast, adding ALA in the presence of PSO normalized embryo fragmentation, cleavage, blastocyst rates, and blastocyst quality compared to controls (P>0.05). Combined treatment with ALA also reduced CC apoptosis, partially recovered CC expansion, abrogated the reduction in MMP in the CCs but not in the oocytes, and reduced BiP and HSP70 expression in CCs, compared with PSO only (P<0.05). In conclusion, ALA supplementation protected oocyte developmental capacity under lipotoxic conditions mainly by protecting cumulus cell viability.

The role of oxidative stress in female infertility and in vitro fertilization.

Infertility problem involves many couples of reproductive age. It has been estimated that in Poland 0.7-1.0 million pairs require treatment, while for more than half of them assisted reproduction is the only recommended and effective method. Infertility affects 13 to 15% of the world's population. A major concern is the age-related decline in female fertility even more that often a decision about pregnancy is taken at later age. Recent studies show that increased production of reactive oxygen species is an important factor in etiopathogenesis of pregnancy and affects female reproduction. It was found that oxidative stress may damage the oocytes and may impair their fertilization capacity. Oxidative stress may also lead to embryo fragmentation and formation of numerous developmental abnormalities, and is regarded to be one of the important reasons of spontaneous and recurrent miscarriage. Moreover, overproduction of reactive oxygen species has a significant impact on the success of in vitro fertilization (IVF).

Polar body transfer restores the developmental potential of oocytes to blastocyst stage in a case of repeated embryo fragmentation.

We aimed to determine the developmental potential of human reconstructed oocytes after polar body genome transfer (PBT) and to report the case of a woman with multiple cycles of severe embryo fragmentation. Fresh and cryopreserved first polar bodies (PB1s) were transferred to enucleated metaphase II oocytes (PB1T), while fresh PB2s were removed from fertilized oocytes and used instead of the female pronucleus in donor zygotes. Reconstructed oocytes underwent intracytoplasmic sperm injection (ICSI) and were cultured to blastocyst. Biopsied trophectoderm cells of PBT-derived blastocysts were screened for chromosomes by next-generation sequencing (NGS). Then, cryopreserved PB1T was carried out in one woman with a history of several cycles of extensive embryo fragmentation, and the blastocysts derived from PB1T were screened for aneuploidy but not transferred to the patient. There were no significant differences in the rates of normal fertilization and blastocyst formation between fresh and cryopreserved PB1T and control oocytes. Of the three fresh and three cryopreserved PB1T-derived blastocysts, two and one blastocysts exhibited normal diploidy respectively. In contrast, 17 PB2 transfers yielded 16 two pronuclei (2PN) zygotes with one normal and one small-sized pronucleus each and no blastocyst formation. In the female patient, 18 oocytes were inseminated by ICSI in the fourth cycle and the PB1s were biopsied. Although the embryos developed from the patient's own oocytes showed severe fragmentation, the oocytes reconstructed after PB1T produced three chromosomally normal blastocysts. Normal blastocysts can develop from human reconstructed oocytes after PB1T. The application of the first PB transfers may be beneficial to patients with a history of poor embryo development and excessive fragmentation.

Clinical outcome of cycles with oocyte degeneration after intracytoplasmic sperm injection

ABSTRACTThere are variant rates of oocyte degeneration after intracytoplasmic sperm injection (ICSI) among different patients. Oocyte degeneration after ICSI may reflect the cohort of oocyte quality and subsequent embryo development capacity and clinical outcome. This retrospective study analyzed 255 cycles with at least one degenerated oocyte after ICSI (degeneration group) and 243 cycles with no degenerated oocytes after ICSI (control group). Basic characteristics like female age, body mass index, duration of infertility, hormone (FSH, LH, E2) levels on day 3 of menses, and primary infertility patient rate were similar between the two groups (p > 0.05). Total dose of gonadotropin and length of stimulation were also similar between the two groups (p > 0.05), but the degeneration group exhibited a more exuberant response to ovarian stimulation as reflected by more oocytes retrieved (p < 0.05). The number of 2PN embryos available and high quality embryos were similar between the two groups (p > 0.05), but the high quality embryo rate, early cleavage embryo rate, and available embryo rate were all statistically lower than the control group (p < 0.05). Embryo developmental kinetics seemed to be disturbed and embryo fragmentation rate increased in the degeneration group (p < 0.05). However, there was no statistical difference in the distribution of graded embryos transferred, and there were no statistical differences in the pregnancy rate, implantation rate, and abortion rate between the two groups (p > 0.05). We deduce that the presence of oocyte degeneration after ICSI may be associated with decreased embryo quality with embryo development kinetics disturbed. However, the clinical outcomes may not be affected if the premise that sufficient high quality degeneration group embryos are available for transfer.

Associations between IVF outcomes and essential trace elements measured in follicular fluid and urine: a pilot study.

A hypothesis-generating pilot study exploring associations between essential trace elements measured in follicular fluid (FF) and urine and in vitro fertilization (IVF) endpoints. We recruited 58 women undergoing IVF between 2007 and 2008, and measured cobalt, chromium, copper, manganese, molybdenum, and zinc in FF (n = 46) and urine (n = 45) by inductively coupled plasma mass spectrometry (ICP-MS). We used multivariable regression models to assess the impact of FF and urine trace elements on IVF outcomes, adjusted for age, body mass index, race, and cigarette smoking. Trace elements were mostly present at lower concentrations in FF than in urine. The average number of oocytes retrieved was positively associated with higher urine cobalt, chromium, copper, and molybdenum concentrations. FF chromium and manganese were negatively associated with the proportion of mature oocytes, yet urine manganese had a positive association. FF zinc was inversely associated with average oocyte fertilization. Urine trace elements were significant positive predictors for the total number of embryos generated. FF copper predicted lower embryo fragmentation while urine copper was associated with higher embryo cell number and urine manganese with higher embryo fragmentation. No associations were detected for implantation, pregnancy, or live birth. Our results suggest the importance of trace elements in both FF and urine for intermediate, although not necessarily clinical, IVF endpoints. The results differed using FF or urine biomarkers of exposure, which may have implications for the design of clinical and epidemiologic investigations. These initial findings will form the basis of a more definitive future study.

Telomeres, Reproductive Aging, and Genomic Instability During Early Development.

Implantation rate decreases and miscarriage rate increases with advancing maternal age. The oocyte must be the locus of reproductive aging because donation of oocytes from younger to older women abrogates the effects of aging on fecundity. Nuclear transfer experiments in a mouse model of reproductive aging show that the reproductive aging phenotype segregates with the nucleus rather than the cytoplasm. A number of factors within the nucleus have been hypothesized to mediate reproductive aging, including disruption of cohesions, reduced chiasma, aneuploidy, disrupted meiotic spindles, and DNA damage caused by chronic exposure to reactive oxygen species. We have proposed telomere attrition as a parsimonious way to explain these diverse effects of aging on oocyte function. Telomeres are repetitive sequences of DNA and associated proteins, which form a loop (t loop) at chromosome ends. Telomeres prevent the blunt end of DNA from triggering a DNA damage response. Previously, we showed that experimental telomere shortening phenocopies reproductive aging in mice. Telomere shortening causes reduced synapsis and chiasma, chromosome fusions, embryo arrest and fragmentation, and abnormal meiotic spindles. Telomere length of polar bodies predicts the fragmentation of human embryos. Telomerase, the reverse transcriptase capable of reconstituting shortened telomeres, is only minimally active in oocytes and preimplantation embryos. Intriguingly, during the first cell cycles following activation, telomeres robustly elongate via a DNA double-strand break mechanism called alternative lengthening of telomeres (ALTs). Alternative lengthening of telomere takes place even in telomerase-null mice. This mechanism of telomere elongation previously had been found only in cancer cells lacking telomerase activity. We propose that ALT elongates telomeres across generations but does so at the cost of extensive genomic instability in preimplantation embryos.

Associations between follicular fluid high density lipoprotein particle components and embryo quality among in vitro fertilization patients.

Follicular redox balance is likely to be important for embryo quality during in vitro fertilization (IVF), and the anti-oxidative high desity lipoprotein (HDL) particle is the sole lipoprotein measured in follicular fluid (FF). Therefore, we investigated FF HDL particle components as predictors of embryo quality during IVF. Two research follicles collected from each participant were individually tracked, and 103 women having at least one developed embryo were included in the analysis. Concentrations of 15 non-cholesterol HDL particle components and 26 HDL-cholesterol (HDL-C) particle size subfractions were determined. Embryo quality was assessed for embryo cell number, embryo fragmentation, and embryo symmetry. Multivariable Poisson regression with a sandwich variance estimator was used to evaluate associations between HDL particle components and embryo quality, adjusted for covariates. Higher γ-tocopherol concentration was associated with less embryo fragmentation (relative risk [RR] = 4.43; 95% confidence interval [CI] 1.78, 11.06), and higher apolipoprotein A-1 concentration was associated with full embryo symmetry (RR = 3.92; 95% CI 1.56, 9.90). Higher concentrations of HDL-C subfractions in the large and medium particle size ranges were associated with poorer embryo quality. FF HDL lipophilic micronutrients and protein components, as well as HDL-C particle size, may be important predictors of embryo quality during IVF.