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The culture medium plays an important role in embryonic development and subsequent pregnancy outcomes of in vitro fertilization (IVF) cycles. The sequential culture media of Vitrolife and Cook are the two most commonly used reagents in China. This study aimed to assess their effects on IVF success rates, obstetric outcomes and neonatal outcomes.This was a retrospective cohort study on 6352 patients undergoing first IVF attempts between January 2018 and December 2019, resulting in 3153 clinical pregnancies, 2646 live births, and 2668 babies. Patients were grouped according to the culture media they used. There were 4680 patients in the Vitrolife group and 1672 patients in the Cook group. The primary outcome measures were maternal and neonatal outcomes. ANOVA and Chi-square tests were used for statistical comparison, and multivariate logistic regression and multivariate general linear model were used to adjust for potential confounders.The rates of live birth, clinical pregnancy, monozygotic twins, miscarriage, and ectopic pregnancy were all comparable between the groups of Vitrolife and Cook. The incidence of placenta previa was higher in the Vitrolife group [4.86 vs. 3.09%, adjusted odds ratio = 2.048 (1.146-3.657)]. The incidence of macrosomia was higher in the Cook group [7.51 vs. 5.39%, adjusted odds ratio = 1.445 (1.010-2.069)].The culture media of Vitrolife and Cook are comparably effective in IVF success. Vitrolife is associated with a higher risk of placenta previa, while Cook is associated with a higher risk of macrosomia.

PURPOSE: To compare morphokinetic parameters in embryos obtained from women with and without endometriosis. METHODS: We evaluated a total of 3471 embryos resulting from 434 oocyte retrievals performed at a single academic center. One thousand seventy-eight embryos were obtained from women affected by endometriosis and 2393 came from unaffected controls. All embryos were cultured in a time-lapse incubator chamber for up to 6 days. IVF cycle outcomes and morphokinetic parameters collected prospectively were retrospectively reviewed. RESULTS: Morphokinetic data suggest that embryo development is impaired in embryos obtained from women with endometriosis (EE). EE were slower to achieve the 2–8 cell stages compared to control embryos (CE) (p

To determine whether glutamine consumption is associated with embryo quality and aneuploidy, a retrospective study was conducted in an in vitro fertilization center. Spent embryo culture media from patients undergoing assisted reproduction treatment and preimplantation genetic testing (PGT) were obtained on day 3 of in vitro culture. Embryo quality was assessed for cell number and fragmentation rate. PGT for aneuploidy was performed using whole genome amplification and DNA sequencing. Glutamine levels in spent embryo culture media were analyzed by gas chromatography–mass spectrometry. The results demonstrated that glutamine was a primary contributor to the classification of the good-quality and poor-quality embryos based on the orthogonal partial least-squares discriminant analysis model. Glutamine consumption in the poor-quality embryos was significantly higher than that in the good-quality embryos (P P

PURPOSE: We aimed to evaluate the link between the GDF9 concentration in day 3 human embryo culture medium and embryo quality and viability. METHODS: Two independent, prospective, observational studies were conducted. In study 1, a total of 280 embryos from 70 patients who obtained at least 4 embryos with 6–10 blastomeres (2 transferable and 2 non-transferable embryos) at day 3 were enrolled. In study 2, a total of 119 embryos from 61 patients (29 fully implanted and 32 non-implanted patients) were enrolled. The corresponding GDF9 concentrations in spent culture medium of embryos were quantified by ELISA assay. The expression pattern of GDF9 in human embryos was investigated using Q-PCR and immunofluorescence. RESULTS: GDF9 mRNA and protein were detected from human oocytes to eight-cell embryos and displayed a slow decreasing trend. In study 1, GDF9 concentration in culture medium is lower for transferable embryos compared with non-transferable embryos (331 pg/mL (quartiles: 442, 664 pg/mL) vs. 518 pg/mL (quartiles: 328, 1086 pg/mL), P

The effects of culture media on DNA methylation process, which is one of the epigenetic mechanisms, have not been clearly elucidated although it is known that in vitro culture conditions alter epigenetic mechanisms. This study was designed to address the question: does embryo culture media approach, sequential or single step, differentially affect DNA methylating enzymes and global DNA methylation. Mouse zygotes were cultured either in single step or sequential culture media until the blastocyst stage and in vivo developed blastocyst were utilized as control. Similarly, GV stage oocytes were in vitro matured either in single step or first step of sequential culture media. In vivo matured MII oocytes were used as control. The expression levels and cellular localization of Dnmt1 and 3a enzymes were analyzed by immunofluorescence and western blot analysis while global DNA methylation was evaluated by immunofluorescence. We found that signal intensities of Dnmt1 and Dnmt3a enzymes were significantly low in embryos or oocytes cultured in sequential media compared to single step media and control, which were comparable amongst themself. Similarly, global DNA methylation level in single step media and control groups was comparable but both was higher than the sequential media. This study demonstrated that composition of culture media may differentially affect DNA methylation levels in mouse embryos and oocytes. Since abnormal DNA methylation may cause aberrant oocyte or embryo development, we think that further studies are needed to test human embryos and oocyte, and to explain molecular mechanisms.

OBJECTIVES: Using time-lapse microscopy, previous research has shown that IVF mouse embryos that cleave earlier at the first division (‘fast’) develop into blastocysts with increased glucose consumption and lower likelihood of post-implantation loss as compared to slower cleaving embryos (‘slow’). Further, metabolomics analysis employing LC–MS conducted on groups of ‘fast’ blastocysts revealed that more aspartate was consumed. With the worldwide adoption of single blastocyst transfer as the standard of care, the need for quantifiable biomarkers of viability, such as metabolism of specific nutrients, would greatly assist in embryo selection for transfer. METHODS: Here we describe the development of a targeted enzymatic assay to quantitate aspartate uptake of single blastocysts. RESULTS: Results demonstrate that the rates of aspartate and glucose consumption were significantly higher in individual ‘fast’ blastocysts. Blastocysts, together with placental and fetal liver tissue collected following transfer, were analysed for the expression of genes involved in aspartate and carbohydrate metabolism. In ‘fast’ blastocysts, expressions of B3gnt5, Slc2a1, Slc2a3, Got1 and Pkm2 were found to be significantly higher. In placental tissue derived from ‘fast’ blastocysts, expression of Slc2a1, Got1 and Pkm2 were significantly higher, while levels of Got1 and Pkm2 were lower in fetal liver tissue compared to tissue from ‘slow’ blastocysts. CONCLUSIONS: Importantly, this study shows that genes regulating aspartate and glucose metabolism were increased in blastocysts that have higher viability, with differences maintained in resultant placentae and fetuses. Consequently, the analysis of aspartate uptake in combination with glucose represents biomarkers of development and may improve embryo selection efficacy and pregnancy rates.

PURPOSE: The few studies that examined the effect of male and/or female features on early embryo development, notably using the time-lapse system (TL), reported conflicting results. This can be explained by the small number of studies using an adapted model. METHODS: We used two original designs to study the female and male effects on embryo development: (1) based on embryos from donor oocytes (TL-DO), and (2) from donor sperm (TL-DS). Firstly, we analyzed the female and male similarities using an ad hoc intraclass correlation coefficient (ICC), then we completed the analysis with a multivariable model to assess the association between both male and female factors, and early embryo kinetics. A total of 572 mature oocytes (TL-DO: 293; TL-DS: 279), fertilized by intracytoplasmic sperm injection (ICSI) and incubated in a TL (Embryoscope®) were included from March 2013 to April 2019; 429 fertilized oocytes (TL-DO: 212; TL-DS: 217) were assessed. The timings of the first 48 h have been analyzed. RESULTS: The similarities in the timings thought to be related to the female component were significant: (ICC in both DO-DS designs respectively: tPB2: 9–18%; tPNa: 16–21%; tPNf: 40–26%; t2: 38–24%; t3: 15–20%; t4: 21–32%). Comparatively, those related to male were lower. Surprisingly after multivariable analyses, no intrinsic female factors were clearly identified. However, in TL-DO design, oligozoospermia was associated with a tendency to longer timings, notably for tPB2 (p = 0.026). CONCLUSION: This study quantifies the role of the oocyte in the first embryo cleavages, but without identified specific female factors. However, it also highlights that sperm may have an early embryonic effect. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10815-021-02336-5.

Tubulin beta eight class VIII (TUBB8) is a subtype of β-tubulin that only exists in primates. TUBB8 mutations have been reported to cause arrest of oocyte maturation and embryonic development. We aim to further investigate the mutational spectrum of TUBB8 and its relevance with female infertility. In our study, infertile patients were recruited, and their basal and clinical characteristics were analyzed. Genomic DNA was extracted from peripheral blood donated by patients. Candidate variants were identified by whole-exome sequencing, selected by relevant criteria, and validated by Sanger sequencing. We found five heterozygous variants: c.C208A(p.P70T), c.T907C(p.C303R), c.G173A(p.R58K), c.G326T(p.G109V), and c.C916T(p.R306C) in TUBB8 among six infertile patients characterized by abnormal phenotypes in oocyte maturation, fertilization, or embryo development. Most of oocytes retrieved from affected individuals were arrested at GV (germinal vesicle) stage and early embryos were arrested at variable stages. In vitro experiments were performed, and the relationship between variant c.G173A(p.R58K), c.C208A(p.P70T), and infertility phenotype was confirmed. We also discussed the possibility about patient II-1 from family 4 is affected by germinal/germline mosaicism. These results expand the kinds of variants and phenotypic spectrum of TUBB8 variants with regard to female infertility.

In the female reproductive tract, oocytes and embryos are in a dark environment, while during the in vitro fertilization (IVF) they are exposed to various visible and invisible lights such as daylight, microscope, and laminar hood fluorescent lights. Studies have shown that light could damage cellular compartments of oocytes and embryos and consequently decrease rates of fertilization, development, and blastocyst formation. However, due to the lack of consensus about the effects of light on the embryos, and subsequently the inability to make definitive decisions regarding the light exposure management to improve IVF results, in the present study, we systematically reviewed the effect of light with different wavelengths and intensities on pre-implantation embryos. The toxic impact of light depends on the wavelength, intensity, and duration of light exposure and also the stage of embryo. Therefore, reducing the observation time of embryos out of the incubator and also using light filters can alleviate the detrimental effect of light in IVF labs.

PURPOSE: To understand the clinical factors associated with embryo survival after vitrification in a cohort of human blastocysts screened by preimplantation genetic testing for aneuploidy (PGT-A). METHODS: Patient demographic, embryo, and cycle characteristics associated with failed euploid blastocyst survival were compared in a cohort of women (n = 6167) who underwent IVF-PGT-A. RESULTS: Compared to those that survived warming, vitrified euploid embryos that failed to survive after warming came from IVF cycles with significantly higher estradiol levels at time of surge (2754.8 ± 1390.2 vs. 2523.1 ± 1190.6 pg/mL, p = 0.03), number of oocytes retrieved (19.6 ± 10.7 vs. 17.5 ± 9.8, p = 0.005), and basal antral follicle count (BAFC) (15.3 ± 8.5 vs. 13.9 ± 7.2, p = 0.05). Euploid embryos were less likely to survive warming if they came from cycles before 2015 (24.6% vs. 13.2%, p

Background During embryonic development, the normality of cleavage and the ploidy state are closely related to the final clinical outcome. At present, many research teams are focusing on the combined application of timelapse (TL) technology and preimplantation genetic testing (PGT) technology, hoping to find a connection between the two aspects of morphodynamics and genes. In the process of embryonic cleavage, there is a common abnormal cleavage pattern called reverse cleavage (RC). RC refers to blastomere fusion and failed cytokinesis. There are very few reports about it. Whether the occurrence of RC affects blastocyst euploidy is even less clear. Whether the RC phenomenon affects the embryonic developmental potential and whether it is related to the embryo ploidy. This is important for clinicians and embryologists. In this study, we used TL to observe whether there was a phenomenon of RC in each biopsy embryo and then combined it with the ploidy state to give an answer, which provided support for the selection strategy of RC embryos. Methods A total of 405 TL-PGT cycles and 1,467 blastocysts were included in the study. All TL data were collected from the Reproductive Medicine Center, Huazhong University of Science and Technology Hospital. Embryos images throughout embryonic development, from post-insemination to day 5 or 6 until biopsy and cryopreservation, were acquired by the Embryoscope Plus TL microscopy system from January 2019 to December 2020. This study investigated the overall incidence of RC during cleavage; the relationship between RC phenomenon and the number of occurrences and ploidy results; the relationship between RC occurrence and blastocyst developmental quality, as well as the dynamics of RC embryos. Results Among the 1,453 blastocysts biopsied, 400 blastocysts showed RC phenomenon at the cleavage stage, and the incidence rate was 25.9%. In euploid, mosaic and aneuploid embryos, the incidence of RC was 27.2%, 26.6%, and 25.0%, respectively. The incidence of RC was similar among these three groups with no significant difference (P > 0.05). The number of RC occurrences was not associated with embryo ploidy status (P > 0.05). In general, the blastocyst quality of the RC + group was lower than that of the RC- group. In the ICM score, the proportion of A score in the RC + group was significantly lower than that in RC- group (P P P Conclusions The chromosomal euploidy of cleavage-stage embryos with RC phenomenon developed to the blastocyst stage was not significantly different from that of cleavage normal blastocysts. Therefore, RC embryos should not be discarded. It is recommended to select and utilize blastocyst culture, which has similar clinical value to normal cleavage embryos.

PURPOSE: Is the presence of cytoplasmic strings (CS) in human blastocysts associated with the probability of clinical pregnancy with fetal heart (CPFH) after transfer. METHODS: This case-control study involved 300 single blastocyst transfers. 150 of these resulted in a CPFH (cases) while 150 did not (controls). All embryos were cultured in Embryoscope+ and AI software (IVY) was used to select the blastocyst with the highest score from the cohort for transfer. An embryologist, blind to the transfer outcome, recorded the CS number, location, and duration of their activity. RESULTS: There was a significant difference in the number of blastocysts that contained CS, with 97.3% of women’s blastocysts resulting in +CPFH containing the CS compared to 88.7% of blastocysts in women who did not have a pregnancy (p = 0.007, OR; 4.67, CI 95% 1.5–14.2). CS appeared 2.4 h earlier in embryo development in the +CPFH group compared to their negative counterparts (p = 0.007). There was a significant difference in the average number of CS/blastocyst with a higher number being present in those that achieved a clinical pregnancy (mean: 6.2, SD 2.9) compared to those that did not (mean: 4.6, SD 3.0) (p ≤ 0.0001). There was a significant increase in the number of vesicles seen traveling along the CS with more seen in the blastocysts resulting in a +CPFH (mean: 4.3 SD 2.1) compared to those in the −CPFH group (mean: 3.1, SD 2.1). CONCLUSION: This study has shown that the presence of cytoplasmic strings in human blastocysts is associated with the probability of clinical pregnancy with fetal heart. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10815-021-02213-1.

Although in vitro culture of human embryos is a crucial step in assisted reproduction, the lack of focused research hampers worldwide standardisation and consistent outcomes. Only 1.2% of research papers published in five leading journals in human reproduction in 2019 focused on in vitro culture conditions, creating the impression that the optimisation process has approached its limits. On the other hand, in vitro culture of mammalian embryos is based on old principles, while there is no consensus on basic issues as density, time, medium change, gas atmosphere and small technical details including the way of drop preparation. This opinion paper aims to highlight and analyse the slow advancement in this field and stimulate research for simple and affordable solutions to meet the current requirements. A possible way for advancement is discussed in detail. Selection of embryos with the highest developmental competence requires individual culture and modification of the widely used "drop under oil" approach. Current use of three-dimensional surfaces instead of large flat bottoms is restricted to time-lapse systems, but these wells are designed for optical clarity, not for the needs of embryos. The size and shape of the original microwells (Well of the Well; WOW) offer a practical and straightforward solution to combine the benefits of communal and individual incubation and improve the overall quality of cultured embryos.

PURPOSE: To determine if euploidy rates and embryo development differ when blastocysts are cultured in CCM or SCM. METHOD: A single-center retrospective observational study was performed from September 2018 to March 2019. Patients [23–46 years] with at least four fresh mature oocytes (MII) without severe male factor infertility were included. Sibling MII were injected and cultured in Global®Total®LP (CCM) or Sage Quinn’s Advantage® Cleavage and Blastocyst media (SCM) under 6% CO(2), 5% O(2), and 89% N(2). Fertilization, cleavage, day (D) 5 blastulation, usable blastocyst (blastocysts biopsied/normally fertilized oocytes), and euploidy rates were recorded. Blastocysts were graded prior to trophectoderm (TE) biopsy on D5, 6, or 7 for genetic testing and mitochondrial DNA (mtDNA) quantification. RESULTS: According to clinical practice, 1452 MII were randomly distributed: 751 in CCM and 701 in SCM. No differences were observed in fertilization and cleavages rates for CCM and SCM (77.4% vs 75.5%, p = 0.429 and 97.6% vs 99.1%, p = 0.094, respectively). Blastulation rate on D5 was higher in CCM (70.6% vs 62.2, p = 0.009); however, usable blastocyst rates were comparable (CCM: 58.3% vs SCM: 56.7%, p = 0.625). From a Poisson regression model adjusted for confounding factors, euploidy rates were not different between media (aOR = 1.18, [0.94–1.48], p = 0.157). Euploid blastocyst’s mtDNA values were similar (CCM: 32.2, [30.5, 34.1] and SCM: 33.5, [31.8, 35.2], p = 0.345) and top-quality blastocysts (AA/BA) were increased in SCM (OR=1.04, [1.00–1.09], p = 0.037). CONCLUSION: Under controlled in vitro conditions, euploidy rates and embryo development are comparable when embryos are cultured in CCM or SCM. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10815-021-02187-0.

PURPOSE: This study aims to know what proportion of culture day 5 pre-blastocyst-stage embryos develop into blastocysts by culture day 6 and what patient and cycle characteristics are associated with delayed blastocyst formation. METHODS: A retrospective observational cohort analysis was performed including a total of 9886 embryos from 1008 IVF cycles in 835 patients, who underwent treatment between January 1, 2016, and December 31, 2018. Autologous fresh in vitro fertilization (IVF) cycles at a single academic center were included in the analysis. Embryos were group-cultured using single-step culture media. Blastulation was defined as the presence of a new blastocyst. Usable blastulation was defined as the presence of a new good or excellent quality, expanded, hatching, or hatched blastocysts. RESULTS: The mean blastulation rate between days 5 and 6 of extended embryo culture was 30.9%. The mean percentage of embryos developing into usable blastocyst-stage embryos was 19.8%. The factors associated with blastulation on day 6 included the total number of embryos and the number of pre-blastocysts on day 5, as well as the use of ICSI. Age, the number of total embryos, those remained in culture and pre-blastocysts, as well as the blastulation rate on day 5 were associated with usable blastulation. CONCLUSION: It is important to know the usable blastocyst development rate between culture days 5 and 6 in order to adequately counsel patients debating whether to proceed with fresh ET on day 5 or forego ET with the expectation that embryos will be biopsied for PGT and/or cryopreserved on culture day 6. Our findings provide evidence to help guide patients in this difficult decision.

PURPOSE: To assess whether the GnRH-agonist or urinary-hCG ovulation triggers affect oocyte competence in a setting entailing vitrified-warmed euploid blastocyst transfer. METHODS: Observational study (April 2013–July 2018) including 2104 patients (1015 and 1089 in the GnRH-a and u-hCG group, respectively) collecting ≥1 cumulus-oocyte-complex (COC) and undergoing ICSI with ejaculated sperm, blastocyst culture, trophectoderm biopsy, comprehensive-chromosome-testing, and vitrified-warmed transfers at a private clinic. The primary outcome measure was the euploid-blastocyst-rate per inseminated oocytes. The secondary outcome measure was the maturation-rate per COCs. Also, the live-birth-rate (LBR) per transfer and the cumulative-live-birth-delivery-rate (CLBdR) among completed cycles were investigated. All data were adjusted for confounders. RESULTS: The generalized-linear-model adjusted for maternal age highlighted no difference in the mean euploid-blastocyst-rate per inseminated oocytes in either group. The LBR per transfer was similar: 44% (n=403/915) and 46% (n=280/608) in GnRH-a and hCG, respectively. On the other hand, a difference was reported regarding the CLBdR per oocyte retrieval among completed cycles, with 42% (n=374/898) and 25% (n=258/1034) in the GnRh-a and u-hCG groups, respectively. Nevertheless, this variance was due to a lower maternal age and higher number of inseminated oocytes in the GnRH-a group, and not imputable to the ovulation trigger itself (multivariate-OR=1.3, 95%CI: 0.9–1.6, adjusted p-value=0.1). CONCLUSION: GnRH-a trigger is a valid alternative to u-hCG in freeze-all cycles, not only for patients at high risk for OHSS. Such strategy might increase the safety and flexibility of controlled-ovarian-stimulation with no impact on oocyte competence and IVF efficacy. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10815-021-02124-1.

In order to maintain pregnancy rates following single embryo transfer, optimisation of embryo culture and selection is vital. Time-lapse monitoring (TLM) has the potential to play a crucial role by providing sequential images of embryo development and minimal disturbance. Therefore, in this study morphometric assessment of blastocyst area and maximum width was performed in order to evaluate if these parameters are associated with pregnancy outcomes in IVF/ICSI cycles. This is a retrospective study of 664 patients who had elective single blastocyst transfer (eSBT). The EmbryoScope drawing tools were used to measure specific variables such as the maximum blastocyst width and blastocyst area. Our results show that women who were pregnant had significantly (P

To present a new day 4 (D4) embryo grading system to assess embryos in frozen–thawed embryo transfer (FET) cycles. A new grading system (grades A–E) was developed from the 2011 ESHRE Istanbul Consensus for D4 embryos in FET cycles. Embryos with complete compaction were classified as grade A; those with partial compaction were assigned as grade B; and those without compaction were classified as grades C, D, and E according to their different blastomere number ratio (BNR; number of embryo blastomeres on D4/number of embryo blastomeres on D3, D4/D3). Embryos with a BNR of ≥ 1.5 were defined as grade C, those with a BNR of ≥ 1.2 and

To determine whether embryo developmental stage or morphological grading can predict live birth rate (LBR) from a single blastocyst in nonbiopsied and biopsied frozen embryo transfer (FET) cycles. This retrospective study included 1336 nonbiopsied and 360 euploid FET cycles. Blastocysts were divided according to developmental stage (day 5 [D5] and day 6 [D6]) and morphology (good quality and low quality). Nonbiopsied cycles in which D5 blastocysts were transferred were associated with a significantly higher LBR than those in the D6 group (48.5 vs. 24.3%; p

PURPOSE: To determine whether maternal age has an impact on monozygotic twinning (MZT) rates in women undergoing single embryo transfer (SET). METHODS: This is a retrospective cohort study analyzed for the incidence of MZT of all clinical pregnancies after a single embryo transfer was carried out between 2014 and 2018. The effect of different assisted reproductive technology (ART) parameters on the incidence of MZT was evaluated. RESULTS: There were a total of 8459 cycles resulting in pregnancy during the study period. Of these pregnancies, 8236 were singletons and 223 were MZT. The preterm birth rate, miscarriage rate, and cesarean section rate were higher in MZT. Birth weight and gestational age at delivery were lower and smaller. In the univariate analysis, the risk of MZT was decreased with frozen embryo transfer (ET). A nonlinear relationship was observed between maternal age and MZT. A negative relationship between maternal age and MZT was observed in the patients’ age ≥ 36 years. CONCLUSION: Advanced maternal age was associated with a lower rate of MZT. A threshold female age of 36 years existed for lower MZT.

To investigate whether there is any detrimental effect of progesterone elevation (PE) on the day of oocyte maturation induction on embryological development potentials. This retrospective single-center cohort study included a total of 1485 individual intracytoplasmic sperm injection (ICSI) cycles between January 2014 and December 2018. Serum progesterone (P) levels were measured on the day of oocyte maturation induction following the GnRH antagonist suppression protocol. Embryological parameters such as maturation, fertilization rate (FR), top-quality embryo (TQE) formation rate per 2PN on day 3, and excellent-quality blastocyst (EQB) formation rate per 2PN on day 5/6 were recorded. The inclusion criteria for women were an age ≤ 37 years, a BMI ≤ 30 kg/m2, and access to a total sperm concentration ≥ 2 million. Groups were stratified according to the serum P levels using the cut-off levels of

Cathecolestrogens are estradiol metabolites produced during folliculogenesis in the mammalian ovary. 2-Hydroxyestradiol (2-OHE2) is one of the most abundant although its role remains unknown. The aim of this study is to investigate whether the presence of 2-OHE2 during the germinal vesicle-to-metaphase II transition affects oocyte meiotic and preimplantation developmental competence. Mouse cumulus-oocyte complexes (COCs), isolated from fully grown antral follicles, were in vitro–matured (IVM) in the presence of 2-OHE2 (0.1, 1, 10 or 100 nM) for 6 or 15 h; then, their meiotic and developmental competence was evaluated using a number of cytological quality markers. With the exception of the highest dose (100 nM), the addition of 2-OHE2 to the IVM medium, did not alter, compared with untreated control, the frequency of oocytes that reached the MII stage. Instead, IVM in the presence of 1 nM 2-OHE2 highly increased the rate of preimplantation development and blastocyst quality. To understand whether this positive effect could be attributed to the events occurring during meiosis resumption, we analysed a number of specific cytological quality markers of the asymmetric division, such as PB-I volume and position, presence and extension of the cortical F-actin cap, meiotic spindle shape and area, and microtubule organisation centre localisation. The results highlighted how the presence of 1 nM 2-OHE2 significantly improved the overall cytological organisation required for a correct asymmetric division. Our results contribute a first step to acknowledge a potential role of this estradiol metabolite during the GV-to-MII transition, contributing to the acquisition of oocytes developmental competence.

The objective of this study was to reduce the negative effects of oxidative stress by decreasing the levels of reactive oxygen species (ROS) through supplementation of the major antioxidants present in elderberries: kuromanin and cyanidin. Oocytes (n = 1150) were supplemented with 100 or 200 μM of kuromanin or cyanidin during maturation, and then evaluated for ROS levels or fertilized and evaluated for penetration, polyspermic penetration, male pronucleus formation, and embryonic development. The ROS levels and incidence of polyspermic penetration were lower (P

PURPOSE: Embryo genotyping in IVF clinics aims to identify aneuploid embryos, and current methodologies rely on costly, invasive and time-consuming approaches such as PGT-A screening. MALDI-ToF-based mass spectral analysis of embryo culture has been demonstrated to be a non-invasive, affordable and accurate technique that is able to capture secretome profiles from embryo culture media extremely quick. Thus, aneuploid embryo genotypes can be distinguished from euploids from these profiles towards the development of novel embryo selection tools. METHODS: A retrospective cohort study, including 292 spent media samples from embryo cultures collected from a single IVF clinic in USA. There were 149 euploid and 165 aneuploid embryos previously analysed by PGT-A next-generation sequencing techniques. Secretome mass spectra of embryos were generated using MALDI-ToF mass spectrometry in the UK. Data was systematically analysed using a fully automated and ultra-fast bioinformatic pipeline developed for the identification of mass spectral signatures. RESULTS: Distinct spectral patterns were found for euploid and aneuploid genotypes in embryo culture media. We identified 12 characteristic peak signatures for euploid and 17 for aneuploid embryos. Data analysis also revealed a high degree of complementarity among regions showing that 22 regions are required to differentiate between genotypes with a sensitivity of 84% and a false positive rate of 18%. CONCLUSION: Ultra-fast and fully automated screening of an embryo genotype is possible based on multiple combinations of specific mass spectral peak signatures. This constitutes a breakthrough towards the implementation of non-invasive and ultra-fast tools for embryo selection immediately prior to transfer.

PURPOSE: To evaluate whether mtDNA content at the blastocyst stage differs between embryos derived from fresh or vitrified sibling oocytes. MATERIAL AND METHODS: A retrospective analysis was performed between March 2017 and September 2018, including 504 blastocysts from 94 couples undergoing preimplantation genetic testing for aneuploidies (PGT-A), using fresh oocytes together with previously vitrified oocytes. Trophectoderm biopsies were performed and subjected to next generation sequencing. RESULTS: On average, 1.8 ± 1.0 oocyte vitrification cycles were performed per patient. Between fresh and vitrified cycles, no difference was observed between the number of fertilized oocytes (5.3 ± 4.2 versus 5.5 ± 3.0). Blastulation rate on day 5 per fertilized oocyte was significantly higher in the fresh group (62% ± 29% versus 44% ± 31%; p

PURPOSE: The aim of this study was to compare the levels of hyperglycosylated human chorionic gonadotropin (hCG-H) secreted from balanced and unbalanced human embryos. METHODS: Single-step culture media samples from 155 good quality embryos, derived from 90 good prognosis patients undergoing intracytoplasmic sperm injection (ICSI), were collected on the fifth day of embryo cultivation. All embryos were tested by next-generation sequencing (NGS) technique. The hCG-H levels in the culture media were evaluated by ELISA kit (Cusabio Biotech, CBS-E15803h) according to the manufacturer’s instructions. Statistical analysis was performed using SPSS v.21 (IBM Corp., Armonk, NY, USA). RESULTS: The NGS analysis revealed that 36% of the embryos (n = 56) were balanced, and 64% of the embryos were unbalanced (n = 99). The presence of hCG-H was confirmed in all embryo culture media samples but was absent in the negative control. In addition, hCG-H concentration was significantly higher in the culture media from unbalanced embryos compared with the balanced ones (0.72 ± 0.30 mIU/ml vs. 0.62 ± 0.12 mIU/ml, p = 0.02, respectively). Furthermore, the mean levels of hCG-H were significantly increased in the samples from embryos with multiple abnormalities. Finally, the highest levels of hCG-H were expressed from embryos with monosomy of chromosome 11 (1.28 ± 0.04 mIU/ml) and those with trisomies of chromosomes 21 (2.23 mIU/ml) and 4 (1.02 ± 0.35 mIU/ml). CONCLUSION: Our results suggest that chromosomal aberrations in human embryos are associated with an increased secretion of hCG-H. However, hCG-H concentration in embryo culture media as a single biomarker is not sufficient for an accurate selection of balanced embryos.

PURPOSE: To investigate the associations of previous pregnancy failures, including implantation failures (IFs), biochemical pregnancy losses (BPLs), and early (EMs) and late miscarriages (LMs), with blastocyst aneuploidy and pregnancy outcomes after PGT-A. METHODS: This study included 792 couples who underwent PGT-A after multiple pregnancy failures. Subgroup analyses were used to compare the blastocyst aneuploidy rate (BAR), implantation rate (IR), early miscarriage rate (EMR), and live birth rate (LBR). Multiple linear and logistic regression models were used to evaluate the associations. The control group comprised couples with ≤ 2 IFs, ≤ 1 BPL, ≤ 1 EM, and no LM. RESULTS: Notably, a history of ≥ 4 IFs was significantly associated with an increase in aneuploid blastocysts (42.86% vs. 33.05%, P = 0.044, B = 10.23 for 4 IFs; 48.80% vs. 33.05%, P = 0.002, B = 14.43 for ≥ 5 IFs). Women with ≥ 4 prior EMs also harbored more aneuploid blastocysts (41.00% vs. 33.05%, P = 0.048; B = 9.23). Compared with the control group, women with ≥ 4 prior EMs had a significantly higher EMR (6.58% vs. 31.11%, P

Purpose To evaluate the relationship between elective blastocyst transfer, freezing strategy, and the abandonment of frozen embryos with a storage time limit of 10 years as specified in the National Assisted Reproduction Act of Taiwan. Methods This two-phase retrospective cohort study was conducted at a single tertiary center, Kaohsiung Chang Gung Memorial Hospital (KCGMH), in 2019. Participants were selected from a data registry containing 4167 fresh IVF cycles, including phase 1 cycles from 1999 to 2009 and phase 2 cycles from 2010 to 2014, at KCGMH. Results In phase 1, embryo abandonment was associated with the production of more mature oocytes and embryos, the freezing of more embryos, young female age, blastocyst transfer, and positive pregnancy results. After adjustment for confounding factors, only positive pregnancy results (adjusted odds ratio [aOR] 4.38, 95% confidence interval [CI] 3.17, 6.04), the freezing of ≥ 2 embryos (aOR 3.68, 95% CI 3.10, 4.38), the production of ≥ 6 embryos (aOR 1.68, 95% CI 1.03, 2.73), and the use blastocyst transfer (aOR 2.46, 95% CI 1.64, 3.69) remained significantly associated with embryo abandonment. The factors associated with embryo abandonment or possible abandonment were similar in phase 2. Conclusion For elective blastocyst stage transfer and a freezing strategy performed according to the Taiwan National Assisted Reproduction Act, a young female age ≤ 35 with positive pregnancy status due to the original IVF treatment, the production of ≥ 6 embryos, and the cryopreservation of ≥ 2 blastocysts may increase the likelihood of abandoning embryos in the future.

PURPOSE: To evaluate clinical outcomes after frozen-thawed blastocyst transfer (TBT) with blastocysts which were derived from different cell numbers on day 3. METHODS: The study included 1444 patients undergoing single autologous frozen-thawed blastocyst transfer cycles, which were allocated to five groups according to the cell numbers on day 3 of the transferred blastocysts: ≤ 6-cell (n = 109), 7-cell (n = 169), 8-cell (n = 811), 9-cell (n = 136), and ≥ 10-cell (n = 219). RESULTS: The LBR of the ≤ 6-cell group was found to be statistically lower than that of the 8-cell group in single TBT cycles which had been transferred with fair quality blastocysts (defined as 4BB according to Gardner’s grading scale) (41.28% vs 55.73%, P = 0.004), while the miscarriage rate was significantly higher for the ≤ 6-cell group compared with the 8-cell group (25.00% vs 13.74%, P = 0.02). No differences were found between the two groups in terms of cPR (P = 0.06). However, for blastocysts categorized as high quality according to Gardner’s classification (defined as 4AA/4AB/4BA), cPR, LBR, and early miscarriage rates did not differ between the two groups (P = 0.76, P = 0.44, P = 0.40, respectively). CONCLUSIONS: When transferring blastocysts, an evaluation of the cleavage stage should be performed along with blastocyst morphology to shorten the time of conceiving. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s10815-019-01664-x) contains supplementary material, which is available to authorized users.

PURPOSE: To compare a single-step medium with a sequential medium on human blastocyst development rates, aneuploidy rates, and clinical outcomes. METHODS: Retrospective cohort study of IVF cycles that used Sage advantage sequential medium (n = 347) and uninterrupted Sage 1-step medium (n = 519) from July 1, 2016, to December 31, 2017, in an academic fertility center. Main outcome measures are blastocyst formation rates per two-pronuclear (2PN) oocyte and aneuploidy rates per biopsy. RESULTS: Of all IVF cycles, single-step medium yielded higher blastocyst formation rate (51.7% vs 43.4%) but higher aneuploidy rate (54.0% vs 45.8%) compared with sequential medium. When stratified by maternal age, women under age 38 had no difference in blastocyst formation (52.2% vs 50.2%) but a higher aneuploidy rate (44.5% vs 36.4%) resulting in a lower number of euploid blastocysts per cycle (2.6 vs 3.3) when using single-step medium compared to sequential medium. In cycles used single-step medium, patients ≥ age 38 had higher blastocyst rate (48.0% vs 33.6%), but no difference in aneuploidy rate (68.8% vs 66.0%) or number of euploid embryos (0.8 vs 1.1). For patients reaching euploid embryo transfer, there was no difference in clinical pregnancy rates, miscarriage rates, or live birth rates between two culture media systems. CONCLUSIONS: Our study demonstrates an increase in aneuploidy in young women whose embryos were cultured in a single-step medium compared to sequential medium. This study highlights the importance of culture conditions on embryo ploidy and the need to stratify by patient age when examining the impact of culture conditions on overall cycle potential. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s10815-019-01621-8) contains supplementary material, which is available to authorized users.

During development, pseudostratified epithelia undergo large scale morphogenetic events associated with increased mechanical stress. Using a variety of genetic and imaging approaches, we uncover that in the mouse E6.5 epiblast, where apical tension is highest, ASPP2 safeguards tissue integrity. It achieves this by preventing the most apical daughter cells from delaminating apically following division events. In this context, ASPP2 maintains the integrity and organisation of the filamentous actin cytoskeleton at apical junctions. ASPP2 is also essential during gastrulation in the primitive streak, in somites and in the head fold region, suggesting that it is required across a wide range of pseudostratified epithelia during morphogenetic events that are accompanied by intense tissue remodelling. Finally, our study also suggests that the interaction between ASPP2 and PP1 is essential to the tumour suppressor function of ASPP2, which may be particularly relevant in the context of tissues that are subject to increased mechanical stress.

Genetic engineering of non-human primates, which are most closely related to humans, has been expected to generate ideal animal models for human genetic diseases. The common marmoset (Callithrix jacchus) is a non-human primate species adequate for the production of genetically modified animals because of their small body size and high reproductive capacity. Autologous embryo transfer (AET) is routinely utilized in assisted reproductive technologies for humans but not for experimental animals. This study has developed a novel method for efficiently producing mutant marmosets using AET and CRISPR/Cas9 systems. The embryos were recovered from oviducts of naturally mated females, injected with Cas9/guide RNA, and transferred into the oviducts of the donors. This AET method can reduce the time for in vitro culture of embryos to less than 30 min. This method uses an embryo donor as the recipient, thus reducing the number of animals and allowing for “Reduction” in the 3R principles of humane experimental technique. Furthermore, this method can utilize nulliparous females as well as parous females. We applied our novel method and generated the 6 marmosets carrying mutations in the fragile X mental retardation 1 (FMR1) gene using only 18 females including 14 nulliparous females.

We aimed to investigate why the incidence of embryos derived from oocytes with no pronuclei (0PN) decreases using time-lapse monitoring (TLM) versus fixed-point assessment in conventional IVF cycles. We analyzed 514 embryos monitored with TLM 6–9 h after insemination and 144 embryos monitored using microscopic assessment 18–21 h after insemination. The primary endpoint of this study was the incidence of 0PN-derived embryos in short insemination followed by TLM. The secondary endpoint was the duration of insemination. As exploratory endpoints, we analyzed the blastulation rate and cryo-warmed blastocyst transfer outcome of embryos with early PN fading, whereby PN disappeared within

Cloning, through somatic cell nuclear transfer (SCNT), has the potential for a large expansion of genetically favorable traits in a population in a relatively short term. In the present study we aimed to produce multiple cloned camels from racing, show and dairy exemplars. We compared several parameters including oocyte source, donor cell and breed differences, transfer methods, embryo formation and pregnancy rates and maintenance following SCNT. We successfully achieved 47 pregnancies, 28 births and 19 cloned offspring who are at present healthy and have developed normally. Here we report cloned camels from surgical embryo transfer and correlate blastocyst formation rates with the ability to achieve pregnancies. We found no difference in the parameters affecting production of clones by camel breed, and show clear differences on oocyte source in cloning outcomes. Taken together we demonstrate that large scale cloning of camels is possible and that further improvements can be achieved.

PURPOSE: The present post hoc analysis aims to study the neonatal data of singletons born from three randomized controlled trials (RCTs) which compared the outcome of day 3 and day 5 transfers. METHODS: Our analysis included 208 liveborn singletons from three existing RCTs (publication dates 2004, 2005, and 2006), 93 children from cleavage-stage transfers and 115 from blastocyst-stage transfers. Vanishing twins were excluded from the analysis. Singleton birthweight was the primary outcome measure. Gestational age and gender of the newborn were accounted for in the multiple regression analysis, along with other confounding factors, such as maternal age, BMI, parity, and smoking behavior. RESULTS: There was no significant difference in gestational age (median, interquartile range) between cleavage-stage transfer (275 days; 267–281) and blastocyst-stage transfer (277 days; 270–281; p = 0.22). Singleton birthweight (median, interquartile range) was not significantly different between cleavage-stage transfer (3330 g; 3020–3610) and blastocyst-stage transfer (3236 g; 2930–3630; p = 0.40), even following multivariable regression analysis to control for potential maternal and newborn confounders. CONCLUSION: The gestational age and birthweight were not significantly different after cleavage-stage and blastocyst-stage transfers. One limitation to be recognized is the age of the data, with original data collection dates from 2001 to 2004. Additionally, the RCTs used for the present analysis have a fairly young age restriction.

PURPOSE: To perform a preliminary exploration of a new embryo rank in clinical practice by combining the embryo chromosome copy number and mitochondrial copy number analysis of DNA extracted from embryo culture medium and blastocoel fluid. METHOD: Eighty-three ICSI embryos from day 2 or day 3 were cultured to day 5 or day 6. Thirty-two blastocysts of 3 cc or above were obtained. Culture medium and blastocoel fluid were collected at 24 h before blastocyst formation. The genomic DNA and mitochondrial DNA (mtDNA) from the culture medium combined with blastocoel fluid and the whole blastocyst were amplified and sequenced by MALBAC-NGS. We compared the chromosomal information generated by the new protocol from the culture medium and the information employed by the whole embryo method. A multivariable linear regression was performed to study the impact of the blastocyst morphological score, chromosomal abnormality, embryo mtDNA copy number, and female age on the culture medium mtDNA copy number. RESULTS: (1) The DNA from 31 blastocysts was successfully amplified, and the successful amplification rate was 96.9% (31/32). The success rate of the amplification of genomic DNA extracted from the culture medium was 87.5% (28/32). (2) There were 18 blastocysts in which the less invasive method and the whole embryo method revealed the same results. The consistency rate was 66.7% (18/27). (3) The culture medium mitochondrial DNA copy number (MCN) had a significantly positive correlation with the blastocyst mitochondrial DNA copy number (P = 0.001), female age (P = 0.012), and blastocyst score (P = 0.014), but there was no obvious correlation with blastocyst chromosome (P = 0.138). CONCLUSIONS: The preliminary exploration result of the less invasive approach for having an embryo rank was not satisfying, which still awaits further long-term evaluation.

PURPOSE: To assess the importance of each blastocyst morphological criteria with pregnancy and perinatal outcomes. METHODS: This single-center retrospective cohort study included blastocyst single embryo transfers (SET) performed between 1/2012–2/2018. Poisson regression was used to evaluate pregnancy outcomes following fresh and cryopreserved embryo transfer (CET) for association with blastocyst expansion, inner cell mass (ICM) quality, and trophectoderm (TE) quality. Among cycles resulting in live birth, associations with preterm birth, small for gestational age (SGA) and large for gestational age (LGA), were evaluated using logistic regression. RESULTS: A total of 1023 fresh and 1222 CET cycles were included, of which 465 (45.1%) fresh and 600 (48.5%) CET cycles resulted in singleton live birth. Clinical pregnancy rates increased with increasing expansion among fresh transfers (p for trend = 0.001) but not CET (p = 0.221), and with TE quality for both fresh and CET cycles (p = 0.005 and

PURPOSE: To determine the developmental competence of fast-cleaving D3 embryos. METHODS: Retrospective study including 4028 embryos from 513 PGT-A cycles performed between July 2014 and June 2017. Embryos were cultured in time-lapse incubators and biopsied at blastocyst stage. Embryos were classified in groups according to the number of cells on D3 (from 2-cell to ≥13 -cell and compacted). A generalized linear mixed model adjusted for confounding factors was performed to assess the chance to give rise to an euploid blastocyst in each group compared with the chance of 8-cell embryos. Implantation and live birth rates were also analyzed. RESULTS: The statistical analysis showed that embryos with 9 to 11 cells had a slightly lower euploid blastocyst rate than 8-cell embryos (OR (95% CI) 0.77 (0.61–0.96)) while embryos with more than 11 cells were found to be just as likely to give rise to an euploid blastocyst as the 8-cell embryos (OR (95% CI) 1.20 (0.92–1.56)). Conversely, slow-cleaving embryos had a significantly lower euploid blastocyst rate than 8-cell embryos (OR (95% CI) 0.31 (0.24–0.39)). Moreover, euploid blastocysts derived from fast-cleaving embryos and from 8-cell embryos exhibit similar live birth rates. No significant differences were found in the chance to give rise a live birth between 8-cell and 9- to 11-cell embryos (OR (95% CI) 1.23 (0.70–2.15)) and > 11-cell embryos (OR (95% CI) 1.09 (0.57–2.09)). CONCLUSIONS: Embryos with more than 11 cells exhibit similar developmental competence to 8-cell embryos. Their poor prognosis should be reconsidered. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s10815-019-01574-y) contains supplementary material, which is available to authorized users.

PURPOSE: This study was to evaluate if spent culture media (SCM) of embryos could be used as a non-invasive tool to achieve aneuploidy screening. Ploidy calls, as well as concordance rates between PGT-A results from trophectoderm (TE) and SCM, were compared. Clinical outcomes of single euploid transfers were also evaluated. METHODS: The study was conducted from March 2017 to June 2018 in a university-based ART center. SCM of day 3 to the day(s) of TE biopsy of all biopsied blastocysts were collected for testing. PGT-A results of SCM were compared with the standard results of TE, with clinical relevance and outcomes examined. RESULTS: NiPGT-A using SCM gave a sensitivity of 81.6%, specificity of 48.3%, positive predictive value of 82.6%, and negative predictive value of 46.7% in ploidy calling. The concordance rates for autosomes and sex determination were 62.1% and 82.4%, respectively. There were 14 single embryo transfer cycles of euploids as determined by TE biopsy. Clinical outcomes not only confirmed 3 false positive results from SCM but also reflected the true ploidy status of the transferred embryo in one case. If ploidy calls were dichotomized without mosaic embryos, the sensitivity and NPV would increase to 91.0% and 66.7% (p = 0.60 and p = 0.25), respectively. CONCLUSIONS: Cell-free DNA found in SCM could provide ploidy information of an embryo as in PGT-A from its TE. Given its potential to reflect the comprehensive chromosomal profile of the whole embryo, more research based on clinical outcomes is required to determine if SCM could be a reliable selection tool in PGT-A. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s10815-019-01517-7) contains supplementary material, which is available to authorized users.

The objective of this study was to determine the effect of breed and equine chorionic gonadotropin (eCG) on ovarian response and in vitro embryo production from young goats. Thirty-one (12 Alpine, 10 Nubian, and 9 Saanen) were randomly assigned into three treatments of eCG (T1, 0 IU ; T2, 500 IU ; and T3, 1000 IU). Alpine goats showed the highest amount and largest size of follicles (P = 0.003). The effect of eCG dose 24 h post application was significant (P < 0.05), and was superior in goats undergoing T2. The aspiration rate of cumulus–oocyte complexes (COC) was 34% (P > 0.05), except for percentage of denuded oocytes, which obtained the highest number (P = 0.003) in the Saanen goats. The same difference was found (P = 0.02) in oocytes grade III in T2 and T3, with 42.5 and 37.9% respectively. In vitro embryo production was 80.0% of IVF/cleavage in the Alpine goats (P = 0.003). Embryo production was the greatest for T2 (69.2% ; P = 0.004). T3 goats had higher percentage of morula stage (66.6% ; P = 0.030). It is concluded that the application of eCG has a significant effect on the ovarian status, and quality and quantity of embryos with a differential response depending on the breed.

In recent years, a growing body of literature has emerged investigating the clinical utility of spent embryo media (SEM) for preimplantation genetic testing for aneuploidy (PGT-A) (Hammond et al. in Fertil Steril. 107(1):220–8, 2017; Xu et al. in Proc Natl Acad Sci USA. 113(42):11907–12, 2016; Shamonki et al. in Fertil Steril. 106(6):1312–8, 2016; Feichtinger et al. in Reprod BioMed Online. 34(6):583–9, 2017; Vera-Rodriguez et al. in Hum Reprod. 33(4):745–56, 2018; Kuznyetsov et al. in PLoS One. 13(5):e0197262, 2018; Ho et al. in Fertil Steril. 110(3):467–75, 2018; Capalbo et al. in Fertil Steril. 110(5):870–9, 2018). Most of these studies have reported moderate success rates, suggesting the need for improvements in sensitivity and specificity. The concordance between spent media and embryo biopsy or whole embryo was reported to be between 30.4 and 90%, with 50–70% correlation being the most representative (Xu et al. in Proc Natl Acad Sci USA. 113(42):11907–12, 2016; Shamonki et al. in Fertil Steril. 106(6):1312–8, 2016; Feichtinger et al. in Reprod BioMed Online. 34(6):583–9, 2017; Vera-Rodriguez et al. in Hum Reprod. 33(4):745–56, 2018; Kuznyetsov et al. in PLoS One. 13(5):e0197262, 2018; Ho et al. in Fertil Steril. 110(3):467–75, 2018). Here, we will analyze all spent media testing strategies including SEM collection methods, whole genome amplification (WGA) strategies, chromosome copy number detection, and bioinformatics analysis tools. We will propose improvements to further increase the accuracy and sensitivity of the assay before bringing PGT-A with SEM into the clinical sphere.

Made available in DSpace on 2019-10-06T02:14:59Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-05-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) PurposeWe first assessed regulation of FGF2 expression in cumulus cells by FSH and oocyte-secreted factors during in vitro maturation (IVM). Then, we tested the hypothesis that FGF2 regulates meiotic progression, cumulus expansion, and apoptosis in cumulus-oocyte complexes (COC) undergoing IVM.MethodsIn vitro maturation of bovine COC was utilized as a model to assess regulation of FGF2 expression by FSH and oocyte-secreted factors (via microsurgical removal of the oocyte), as well as effects of graded doses of FGF2 on meiotic progression, degree of cumulus expansion, dissociation of cumulus cells, and cumulus cells apoptosis. Expression of genes regulating functional endpoints altered by FGF2 treatment was assessed in cumulus cells by real-time PCR. Cultures were replicated 4-5 times and effects of treatments were tested by ANOVA.ResultsFGF2 mRNA expression was increased by FSH and oocyte-secreted factors during IVM. Addition of FGF2 to the IVM medium advanced meiosis resumption, decreased the ease with which cumulus cells were dissociated, and inhibited cumulus cells apoptosis. Decreased cumulus dissociation was accompanied by decreased expression of TNFAIP6.ConclusionsThis is the first study showing that FGF2 expression is regulated by the oocyte in cumulus cells. Moreover, we report novel effects of FGF2 on cumulus cell survival and extracellular matrix (ECM) quality during IVM that may favor acquisition of developmental competence and suggest physiological roles during the final steps of COC differentiation. Univ Estadual Paulista, Dept Fisiol, Inst Biociencias, BR-18618970 Sao Paulo, Brazil Univ Montreal, Fac Med Vet, Ctr Rech Reprod & Fertilite, St Hyacinthe, PQ J2S 7C6, Canada Univ Estadual Paulista, Dept Fisiol, Inst Biociencias, BR-18618970 Sao Paulo, Brazil FAPESP: 12/06417-8 CNPq: 312521/2014-6

To investigate the developmental potential and clinical value of embryos with abnormal cleavage rate, a retrospective analysis was performed on 66 635 2-prokaryotic (2PN) and 1-pronuclear (1PN) embryos. The embryos were given conventionally in vitro fertilization (IVF) treatment and continuously cultured on the day 3 (D3) at the Reproductive Medicine Center, Tongji Medical College, Huazhong University of Science and Technology from January 2016 to December 2017. The embryos were separated into the day-2 (D2) undivided group with 106 cases, the arrested development group with 3482 cases, the blastomere reduction group with 541 cases, and the control group with 62 506 cases, respectively. The blastocyst utilization rates of these three abnormal groups were 2.83%, 10.86% and 6.84%, respectively, which were significantly different from that in control group (39.46%). Furthermore, 2 cases of anabiosis and 1 case of live birth were found in D2 undivided group. In arrested development group, there were 55 cases of anabiosis, 11 cases of clinical pregnancy in single-embryo transplantation (including 6 cases of live birth), and 25 cases of clinical pregnancy in combination with one normal embryo transplantation (including 23 cases of live births and 15 cases of dizygotic twins under B-ultrasound). There were 13 case of anabiosis in blastomere reduction group: there was 1 case of single embryo transplantation and clinical pregnancy was obtained; there were also 6 cases of clinical pregnancy in combination with one single normal embryo transplantation (including 5 cases of live births and 2 cases of dizygotic twins under B-ultrasound). In conclusion, embryos with abnormal cleavage rate still have the potential to continue to develop, and have certain blastocyst utilization rate and live birth.

PURPOSE: To demonstrate whether the standard morphokinetic markers used for embryo selection have a similar relationship to blastocyst formation and implantation in two large clinical data sets. METHODS: This is a retrospective cohort analysis striving to answer two distinct questions utilizing data sets from two large IVF clinics. Blastocysts (BL) and implanted blastocysts (I) in both clinics, IVI-Valencia (BL = 11,414, I = 479) and WMC (BL = 15,902; I = 337), were cultured in a time-lapse system (EmbryoScope, Vitrolife, Sweden). The study was designed to assess the relationship between early morphokinetic hallmarks and BL development, with a secondary analysis of implantation rates following single-embryo day 3 and day 5 transfers. RESULTS: We performed a detailed graphical analysis for t3, t5, duration of the second cell cycle (cc2) (t3-t2), and the ratio (t5-t3)/(t5-t2). The t5 timing was not affected between the clinics. However, Weill Cornell Medicine’s (WCM) proportions were significantly affected by having BL vs. not. A significant decrease of blastocysts with longer t5 in WCM data, while t5 was more informative in the IVI data set for the implantation rate. CONCLUSIONS: Morphokinetic intervals for early cleavages were distributed differently between the clinics. Incorporation of embryo-selection algorithms depends on the individual clinic’s selected developmental hallmarks, all of which must be validated before incorporation into clinical practice. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s10815-018-1396-x) contains supplementary material, which is available to authorized users.

This paper aims to investigate the efficacy of IVF with preimplantation genetic testing for aneuploidy (PGT-A), using only best-scoring blastocysts from young (≤ 35 years) infertile patients undergoing single blastocyst frozen embryo transfers (FET). In this randomized controlled trial (RCT) registered 29 March 2017, 302 infertile patient-couples eligible to participate underwent autologous ICSI blastocyst freeze-all cycles. Two-hundred and twenty patient-couples satisfied the inclusion criteria (i.e., female age ≤ 35 years, two-day 5 ≥ 2BB blastocysts) and were randomized to either the PGT-A (PGT-A group, n = 109) selection arm or morphology score (morphology group, n = 111) selection arm. In both arms, the highest ranking (by morphological score) blastocysts were selected for FET. Of the 109 best-scoring blastocysts that underwent PGT-A, 80 were predicted to be euploid (73.4%) and were transferred in FET (euploid subgroup). There was no statistical difference in LB rate between the euploid subgroup and morphology group (56.3% vs 58.6%, odds ratio 0.91 (95% CI 0.51–1.63), p = 0.750). In a multiple logistic regression, the transfer of euploid blastocysts was not found to be a significant predictor of LB when adjusting for female age, infertility duration, antral follicle count, and blastocyst quality, with the independent odds expressed as 0.91 (95% CI 0.50–1.66, p = 0.760). In young (≤ 35 years) infertile patients with at least two ≥ 2BB blastocysts, PGT-A blastocyst selection does not result in an enhanced LB rate, with the evidence suggesting that the effectivity of PGT-A may be limited by the effectivity of TE biopsy. Trial registration: ClinicalTrials.gov ID: NCT03095053.

Background High-quality single blastocyst transfer (SBT) is increasingly recommended to patients because of its acceptable pregnancy outcomes and significantly reduced multiple pregnancy rate compared to double blastocyst transfer (DBT). However, there is no consensus on whether this transfer strategy is also suitable for poor-quality blastocysts. Moreover, the effect of the development speed of poor-quality blastocysts on pregnancy outcomes has been controversial. Therefore, this study aimed to explore the effects of blastocyst development speed and morphology on pregnancy and neonatal outcomes during the frozen embryo transfer (FET) cycle of poor-quality blastocysts and to ultimately provide references for clinical transfer strategies. Methods A total of 2,038 FET cycles of poor-quality blastocysts from patients 40 years old or less were included from January 2014 to December 2019 and divided based on the blastocyst development speed and number of embryos transferred: the D5-SBT (n = 476), D5-DBT (n = 365), D6-SBT (n = 730), and D6-DBT (n = 467) groups. The SBT group was further divided based on embryo morphology: D5-AC/BC (n = 407), D5-CA/CB (n = 69), D6-AC/BC (n = 580), and D6-CA /CB (n = 150). Results When blastocysts reach the same development speed, the live birth and multiple pregnancy rates of DBT were significantly higher than those of SBT. Moreover, there was no statistical difference in the rates of early miscarriage and live birth between the AC/BC and CA/CB groups. When patients in the SBT group were stratified by blastocyst development speed, the rates of clinical pregnancy (42.44 % vs. 20.82 %) and live birth (32.35 % vs. 14.25 %) of D5-SBT group were significantly higher than those of D6-SBT group. Furthermore, for blastocysts in the same morphology group (AC/BC or CA/CA group), the rates of clinical pregnancy and live birth in the D5 group were also significantly higher than those of D6 group. Conclusions For poor-quality D5 blastocysts, SBT can be recommended to patients because of acceptable pregnancy outcomes and significantly reduced multiple pregnancy rate compared with DBT. For poor-quality D6, the DBT strategy is recommended to patients to improve pregnancy outcomes. When blastocysts reach the same development speed, the transfer strategy of selecting blastocyst with inner cell mass “C” or blastocyst with trophectoderm “C” does not affect the pregnancy and neonatal outcomes.

It remained unknown whether HDAC6 affected the histone deacetylation of in vitro maturation oocytes and the reprogramming of nuclear transplantation in pig. Our results indicated that HDAC6 specific inhibition did not affect overall HDAC activity and meiosis process, which increased histone H3K9/K14 and H4K8 acetylation of porcine in vitro maturation oocytes and pseudo-pronucleus embryos. HDAC6 inhibition also significantly enhanced the cleavage and blastocyst of nuclear transfer embryos (0.81 ± 0.12 vs. 0.68 ± 0.12 and 0.46 ± 0.19; 0.73 ± 0.13 vs. 0.63 ± 0.18 and 0.40 ± 0.16, P

Introduction The field of assisted reproductive technology (ART) has significantly advanced; however, morphological evaluation remains as the chosen method of assessment of embryo quality. Objective We aimed to examine metabolic changes in embryo culture medium to develop a non-invasive method for evaluation of embryo quality. Methods We performed metabolic analysis of culture medium obtained from a single blastocyst cultured for freezing. Results In total, 187 (39.8%) of the 469 detectable organic acid metabolites were identified. A significant change (p Conclusion The quantification of metabolism in human embryos may assist in identification and selection of good-quality embryos with high rates of survival before freezing and implantation in conjunction with morphological classification. This may help to identify embryos with high rates of survival.

Background The in vitro maturation (IVM) technique has physical and financial benefits, but a lower efficiency and outcome that is still unclear whether it is related to polycystic ovary syndrome (PCOS) itself or the IVM procedure. In this study, we analyzed the clinical and laboratory outcomes of an optimized IVM protocol in patients with and without PCOS. We also discussed the possible reasons for early embryo arrest in the IVM cycle. Methods This prospective study involved 58 PCOS patients and 56 non-PCOS patients who underwent mild stimulated IVF combined IVM (IVF/M) cycles. The clinical and laboratory outcomes were compared between the two groups. Also, metaphase II (MII) oocytes were obtained after IVM from the two groups, and in vivo MII oocytes randomly collected from IVF patients were examined for mitochondrial function using a laser scanning confocal microscope (LSCM). The aneuploidy rate for arrested cleavage embryos from IVM and IVF oocytes were screened using Next Generation Sequencing (NGS). Results Mildly stimulated IVF/M resulted in cumulative clinical pregnancy and implantation rates (40.2, 28.7% in the PCOS group vs. 41.9, 36% in the non-PCOS group), respectively. The blastocyst formation rates were comparable (28% vs. 28.2%) in PCOS and non-PCOS groups, respectively. Using LSCM, there was a significant decrease in the mitochondrial membrane potential of IVM oocytes compared with the control IVF oocytes (P Conclusions The mildly stimulated IVF/M protocol produced acceptable clinical outcomes in PCOS and non-PCOS patients. IVM itself rather than the PCOS condition adversely affected the embryo development through its effect on mitochondrial function, which appeared to be a possible cause for the embryo arrest in the IVM cycles rather than chromosomal aneuploidy.

Understanding the biological function of amyloid beta (Aβ) precursor protein (APP) beyond its role in Alzheimer’s disease is emerging. Yet, its function during embryonic development is poorly understood. The zebrafish APP homologue, Appb, is strongly expressed during early development but thus far has only been studied via morpholino-mediated knockdown. Zebrafish enables analysis of cellular processes in an ontogenic context, which is limited in many other vertebrates. We characterized zebrafish carrying a homozygous mutation that introduces a premature stop in exon 2 of the appb gene. We report that appb mutants are significantly smaller until 2dpf and display perturbed enveloping layer (EVL) integrity and cell protrusions at the blastula stage.Moreover, appb mutants surviving beyond 48 hpf exhibited no behavioral defects at 6 dpf and developed into healthy and fertile adults. The expression of the app-family members, appa and aplp2, was found to be altered in appb mutants. Taken together, we show that appb orchestrates the initial development by supporting the integrity of the EVL, likely by mediating cell adhesion properties. The loss of Appb might be compensated for by other app family members to be able to implement continued normal development.