Swim-up as a strategy for isolation of spermatozoa without viral incorporation in men with chronic hepatitis B: A pilot study.

Hepatitis B virus (HBV) incorporates into spermatozoa which raises safety concerns about paternofetal transmission performing intracytoplasmatic sperm injection (ICSI) in men with chronic hepatitis B (cHB). HBV reduces sperm cell motility, assuming spermatozoa with highest motility are least HBV-incorporated. This study investigates an ICSI preparation technique (swim-up) to isolate most motile spermatozoa in order to select HBV-free spermatozoa. Semen and blood samples were collected from four patients with cHB. Spermatozoa were incubated in trajectories of gamete medium to create non-motile, motile/non-progressive and motile/progressive fractions. After DNA-extraction, HBV DNA loads were determined in every fraction. Participants (mean age 31) were HBsAg+(4/4), anti-HBc+(4/4) and HBV DNA+(2/4). They were treated (3/4) with entecavir(1/4) or tenofovir (2/4) and had no adverse sperm parameters(3/4). CRP-gene was detected in 95/96 sample fractions, proving successful DNA-extraction. HBV DNA was detected in none of the sample fractions, except for the motile, non-progressive fraction of one patient (HBeAg+, HBV DNA+). Since no HBV DNA was detected in progressive fractions, this study suggests swim-up a successful strategy to select HBV-free spermatozoa. Since all but one fraction was HBV DNA-negative, this study also suggests that patients with well-controlled disease have no HBV-contaminated sample fractions. This study encourages evaluation of guidelines restricting reproductive possibilities in men with cHB.

Endometrial stromal cell proteome mapping in repeated implantation failure and recurrent pregnancy loss cases and fertile women.

RESEARCH QUESTION: Are there proteomic differences between endometrial stromal cells of repeated implantation failure (RIF), recurrent pregnancy loss (RPL) and normal fertile women, and is there differential protein expression upon decidualization? DESIGN: This exploratory study investigated the proteome of in-vitro cultured endometrial stromal cells of women with RIF (n = 4), women with RPL (n = 3) and normal fertile women (n = 4), comparing day 0 with 5 days of decidualization. Total proteins extracted from cell lysates were analysed by high-definition mass spectrometry. Data analysis was performed using significance analysis of microarray in R (P < 0.05; false discovery rate [FDR] 10%). RESULTS: In the RIF group, ANXA6, PSMC5 and FSCN1 were up-regulated (1.9-fold, 2.5-fold and 1.9-fold, respectively), whereas PBXIP1 was down-regulated (7.7-fold) upon decidualization. In the RPL group, RPS25 and ACADVL were down-regulated (1.9-fold and 2.4-fold, respectively; FDR 10%) between the non-decidualized and the decidualized samples. In the normal fertile group VIM and RPL23A were down-regulated (1.9-fold and 2.4-fold, respectively). Comparing ratios of expression of decidualized over non-decidualized samples in the different groups revealed six differentially expressed proteins: DUX4L2, CNPY4, PDE7A, CTSK, PCBP2 and PSMD4. Comparison of RPL versus normal fertile in the decidualized condition revealed serotransferrin to be differentially expressed. The changes in expression levels for serotransferrin, ANX6, ACDVL and VIM were confirmed by western blot. CONCLUSIONS: Results show a varying response of endometrial stromal cells in distinct clinical groups (RIF, RPL and normal fertile) upon in-vitro decidualization. Serotransferrin could serve as a marker for the aberrant decidualization process in RPL.

Patients with a high proportion of immature and meiotically resistant oocytes experience defective nuclear oocyte maturation patterns and impaired pregnancy outcomes.

Patients presenting with abnormally high numbers of immature oocytes at retrieval are more likely to exhibit maturation resistant oocytes. However, the clinical relevance of such events remains unknown. We investigated nuclear maturation competence of immature oocytes from patients showing >40% of collected immature oocytes (Study group) and Controls, in which a normal number of mature oocytes (≥60%) was retrieved. Following in-vitro culture, oocytes were classified as maturation resistant or in-vitro matured (IVM). Treatment outcomes were evaluated in Study and Control groups based on presence of maturation resistant oocytes. Overall, similarly high spindle and chromosome abnormality rates were observed in maturation resistant oocytes from both Study and Control groups. IVM oocytes from the Study group revealed significantly higher percentages of misaligned chromosomes compared with Controls (P < 0.05). Remarkably, Study group patients with at least one maturation resistant oocyte showed significantly reduced cumulative pregnancy and live birth rates compared with Control group maturation resistant patients (P < 0.05). When further investigating the aetiology, a maturation resistant mouse model revealed defective Ca2+ signalling of maturation resistant oocytes at germinal vesicular breakdown and parthenogenetic activation. In conclusion, appropriate treatment strategies, including clinical utilization of IVM oocytes from Study group patients, warrant further investigation.

Ovarian tissue cryopreservation in female-to-male transgender people: insights into ovarian histology and physiology after prolonged androgen treatment.

Female-to-male transgender people (trans men) are faced with the risk of losing their reproductive potential owing to gender-affirming hormone treatment and genital reconstructive surgery. This observational, prospective cohort study investigates the effect of prolonged androgen therapy on their ovarian histology and fertility preservation perspectives. Hormone serum levels, ovarian histology and cumulus-oocyte complexes (COC) of 40 trans men were analysed at the moment of hysterectomy with bilateral oophorectomy in the context of genital reconstructive surgery after testosterone treatment (58.18 ± 26.57 weeks). In the cortex, most follicles were primordial (68.52% total follicle count) compared with 20.26% intermediate and 10.74%primary follicles. Few secondary follicles (0.46%) and a single antral follicle were found in the sections analysed. In total, 1313 COC were retrieved from the medulla of 35 patients (37.51 ± 33.58 COC per patient). Anti-Müllerian hormone serum levels were significantly correlated with number of COC (Rs 0.787, P < 0.001). After 48 h in-vitro maturation, 34.30% metaphase II oocytes were obtained, with 87.10% having a normal spindle structure. In conclusion, the cortical follicle distribution in trans men, after more than a year of testosterone treatment, seems to be surprisingly normal. This work confirms the presence and in-vitro maturation potential of cumulus-oocyte complexes.

Alternative Routes to Induce Naïve Pluripotency in Human Embryonic Stem Cells.

Human embryonic stem cells (hESCs) closely resemble mouse epiblast stem cells exhibiting primed pluripotency unlike mouse ESCs (mESCs), which acquire a naïve pluripotent state. Efforts have been made to trigger naïve pluripotency in hESCs for subsequent unbiased lineage-specific differentiation, a common conundrum faced by primed pluripotent hESCs due to heterogeneity in gene expression existing within and between hESC lines. This required either ectopic expression of naïve genes such as NANOG and KLF2 or inclusion of multiple pluripotency-associated factors. We report here a novel combination of small molecules and growth factors in culture medium (2i/LIF/basic fibroblast growth factor + Ascorbic Acid + Forskolin) facilitating rapid induction of transgene-free naïve pluripotency in hESCs, as well as in mESCs, which has not been shown earlier. The converted naïve hESCs survived long-term single-cell passaging, maintained a normal karyotype, upregulated naïve pluripotency genes, and exhibited dependence on signaling pathways similar to naïve mESCs. Moreover, they undergo global DNA demethylation and show a distinctive long noncoding RNA profile. We propose that in our medium, the FGF signaling pathway via PI3K/AKT/mTORC induced the conversion of primed hESCs toward naïve pluripotency. Collectively, we demonstrate an alternate route to capture naïve pluripotency in hESCs that is fast, reproducible, supports naïve mESC derivation, and allows efficient differentiation.

Follicles of various maturation stages react differently to enzymatic isolation: a comparison of different isolation protocols.

Isolation of human follicles is based on digestion of the tissue by combinations of enzymes. Follicle vitality and morphology are often based on the analysis of pooled follicles of different maturation stages. Information is therefore lacking on the effect of the isolation protocol to individual follicles of different maturation stages. A study was conducted using five protocols combining different enzymes and varying concentrations. Isolated follicles were classified according to their maturation stages, counted and characterized for vitality, morphology, early apoptosis and organization of transzonal projections. No statistical differences were found between the protocols when outcome parameters were analysed on a pool of follicles regardless of their maturation status. Differences were observed in quality when the follicles were analysed separately according to their maturation status. Combining morphologic characteristics and vitality, both Liberase DH and Liberase TM combined with collagenase IV were better at isolating high-quality primordial follicles, compared with collagenase IV. No statistical difference between the isolation protocols was found for primary follicles. If only high-quality isolated secondary follicles are needed, collagenase IV is found to be most advantageous. Follicles of different maturation stages react differently when enzymatic isolation protocols are compared.

Sphingosine-1-phosphate prevents chemotherapy-induced human primordial follicle death.

STUDY QUESTION: Can Sphingosine-1-phosphate (S1P), a ceramide-induced death pathway inhibitor, prevent cyclophosphamide (Cy) or doxorubicin (Doxo) induced apoptotic follicle death in human ovarian xenografts? SUMMARY ANSWER: S1P can block human apoptotic follicle death induced by both drugs, which have differing mechanisms of cytotoxicity. WHAT IS KNOWN ALREADY: S1P has been shown to decrease the impact of chemotherapy and radiation on germinal vesicle oocytes in animal studies but no human translational data exist. STUDY DESIGN, SIZE, DURATION: Experimental human ovarian xenografting to test the in vivo protective effect of S1P on primordial follicle survival in the chemotherapy setting. The data were validated by assessing the same protective effect in the ovaries of xenografted mice in parallel. PARTICIPANTS/MATERIALS, SETTING, METHODS: Xenografted mice were treated with Cy (75 mg/kg), Cy+S1P (200 µM), Doxo (10 mg/kg), Doxo+S1P or vehicle only (Control). S1P was administered via continuous infusion using a mini-osmotic pump beginning 24 h prior to and ending 72 h post-chemotherapy. Grafts were then recovered and stained with anti-caspase 3 antibody for the detection of apoptosis in primordial follicles. The percentage of apoptotic to total primordial follicles was calculated in each group. MAIN RESULTS AND THE ROLE OF CHANCE: Both Cy and Doxo resulted in a significant increase in apoptotic follicle death in human ovarian xenografts compared with controls (62.0 ± 3.9% versus 25.7 ± 7.4%, P < 0.01 and 76.7 ± 7.4% versus 25.7 ± 7.4%, P < 0.01, respectively). This chemotherapy-induced apoptotic death was reduced both in the Cy+S1P (32.7 ± 4.4%, P < 0.01) and the Doxo+S1P group (27.1 ± 7.6%, P < 0.01) compared with Cy and Doxo groups, respectively. In the Doxo+S1P and Cy+S1P groups, the percentages of apoptotic follicles were similar to those of vehicle-treated controls (P > 0.05). The findings from the ovaries of the severe combined immunodeficient mice mirrored the findings with human tissue. LIMITATIONS, REASONS FOR CAUTION: The functionality of the rescued human ovarian follicles needs to be evaluated in future studies though the studies in rodents showed that rescued oocytes can result in healthy offspring. In addition, the impact of S1P on cancer cells should be further studied. WIDER IMPLICATIONS OF THE FINDINGS: S1P and its future analogs hold promise for preserving fertility by pharmacological means for patients undergoing chemotherapy. STUDY FUNDING/COMPETING INTEREST(S): This research is supported by NIH's NICHD and NCI (5R01HD053112-06 and 5R21HD061259-02) and the Flemish Foundation for Scientific Research (FWO-Vlaanderen, grant number FWO G0.065.11N10). The authors have no conflicts of interest to disclose.

A systematic analysis of the suitability of preimplantation genetic diagnosis for mitochondrial diseases in a heteroplasmic mitochondrial mouse model.

STUDY QUESTION: What is the reliability of preimplantation genetic diagnosis (PGD) based on polar body (PB), blastomere or trophectoderm (TE) analysis in a heteroplasmic mitochondrial mouse model? SUMMARY ANSWER: The reliability of PGD to determine the level of mitochondrial DNA (mtDNA) heteroplasmy is questionable based on either the first or second PB analysis; however, PGD based on blastomere or TE analysis seems more reliable. WHAT IS KNOWN ALREADY: PGD has been suggested as a technique to determine the level of mtDNA heteroplasmy in oocytes and embryos to avoid the transmission of heritable mtDNA disorders. A strong correlation between first PBs and oocytes and between second PBs and zygotes was reported in mice but is controversial in humans. So far, the levels of mtDNA heteroplasmy in first PBs, second PBs and their corresponding oocytes, zygotes and blastomeres, TE and blastocysts have not been analysed within the same embryo. STUDY DESIGN, SIZE AND DURATION: We explored the suitability of PGD by comparing the level of mtDNA heteroplasmy between first PBs and metaphase II (MII) oocytes (n = 33), between first PBs, second PBs and zygotes (n = 30), and between first PBs, second PBs and their corresponding blastomeres of 2- (n = 10), 4- (n = 10) and 8-cell embryos (n = 11). Levels of mtDNA heteroplasmy in second PBs (n = 20), single blastomeres from 8-cell embryos (n = 20), TE (n = 20) and blastocysts (n = 20) were also compared. PARTICIPANTS/MATERIALS, SETTING, METHODS: Heteroplasmic mice (BALB/cOlaHsd), containing mtDNA mixtures of BALB/cByJ and NZB/OlaHsd, were used in this study. The first PBs were biopsied from in vivo matured MII oocytes. The ooplasm was then subjected to ICSI. After fertilization, second PBs were biopsied and zygotes were cultured to recover individual blastomeres from 2-, 4- and 8-cell embryos. Similarly, second PBs were biopsied from in vivo fertilized zygotes and single blastomeres were biopsied from 8-cell stage embryos. The remaining embryo was cultured until the blastocyst stage to isolate TE cells. Polymerase chain reaction followed by restriction fragment length polymorphism was performed to measure the level of mtDNA heteroplasmy in individual samples. MAIN RESULTS AND THE ROLE OF CHANCE: Modest correlations and wide prediction interval [PI at 95% confidence interval (CI)] were observed in the level of mtDNA heteroplasmy between first PBs and their corresponding MII oocytes (r(2) = 0.56; PI = 45.96%) and zygotes (r(2) = 0.69; PI = 37.07%). The modest correlations and wide PI were observed between second PBs and their corresponding zygotes (r(2) = 0.65; PI = 39.69%), single blastomeres (r(2) = 0.42; PI = 48.04%), TE (r(2) = 0.26; PI = 54.79%) and whole blastocysts (r(2) = 0.40; PI = 57.48%). A strong correlation with a narrow PI was observed among individual blastomeres of 2-, 4- and 8-cell stage embryos (r(2) = 0.92; PI = 11.73%, r(2) = 0.86; PI = 18.85% and r(2) = 0.85; PI = 21.42%, respectively), and also between TE and whole blastocysts (r(2) = 0.90; PI = 23.58%). Moreover, single blastomeres from 8-cell stage embryos showed a close correlation and an intermediate PI with corresponding TE cells (r(2) = 0.81; PI = 28.15%) and blastocysts (r(2) = 0.76; PI = 36.43%). LIMITATIONS, REASONS FOR CAUTION: These results in a heteroplasmic mitochondrial mouse model should be further verified in patients with mtDNA disorders to explore the reliability of PGD. WIDER IMPLICATIONS OF THE FINDINGS: To avoid the transmission of heritable mtDNA disorders, PGD techniques should accurately determine the level of heteroplasmy in biopsied cells faithfully representing the heteroplasmic load in oocytes and preimplantation embryos. Unlike previous PGD studies in mice, our results accord with PGD results for mitochondrial disorders in humans, and question the reliability of PGD using different stages of embryonic development. TRIAL REGISTRATION NUMBER: Not applicable.

Treatment of human embryos with the TGFß inhibitor SB431542 increases epiblast proliferation and permits successful human embryonic stem cell derivation.

STUDY QUESTION: Is there an effect of the TGFß inhibitor SB431542 (SB) on the epiblast compartment of human blastocysts, and does it affect subsequent human embryonic stem cell (hESC) derivation? SUMMARY ANSWER: SB increases the mean number of NANOG-positive cells in the inner cell mass (ICM), and allows for subsequent hESC derivation. WHAT IS KNOWN ALREADY: It is known that inhibition of TGFß by SB has a positive effect on mouse ESC self-renewal, while active TGFß signalling is needed for self-renewal of primed ESC. STUDY DESIGN, SIZE, DURATION: From December 2011 until March 2012, 263 donated spare embryos were used from patients who had undergone IVF/ICSI in our centre. PARTICIPANTS/MATERIALS, SETTING, METHODS: Donated human embryos were cultured in the presence of SB or Activin A, and immunocytochemistry was performed on Day 6 blastocysts for NANOG and GATA6. Moreover, blastocysts were used for the derivation of hESC, with or without exposure to SB. MAIN RESULTS AND THE ROLE OF CHANCE: Immunocytochemistry revealed a significantly higher number of NANOG-positive ICM cells in the SB group compared with the control (12.0 ± 5.9 versus 6.1 ± 4.7), while no difference was observed in the Activin A group compared with other groups (6.7 ± 3.7). The number of GATA6-positive ICM cells did not differ between the SB, Activin A and control group (8.8 ± 4.3, 8.0 ± 4.6 and 7.2 ± 4.0, respectively). Blocking TGFß signalling did not prevent subsequent hESC line derivation. LIMITATIONS, REASONS FOR CAUTION: The number of human blastocysts available for this study was too low to reveal if the observed increase in NANOG-positive epiblast cells after exposure to SB affected the efficiency of hESC derivation (12.5% compared with 16.7%). WIDER IMPLICATIONS OF THE FINDINGS: This work can contribute to the derivation of naive hESC lines in the future. STUDY FUNDING/COMPETING INTEREST(S): M.V.d.J. is holder of a Ph.D. grant of the Agency for Innovation by Science and Technology (IWT, grant number SB093128), Belgium. G.D. and this research are supported by the Research Foundation Flanders (FWO), grant number FWO-3G062910) and a Concerted Research Actions funding from BOF (Bijzonder Onderzoeksfonds University Ghent, grant number BOF GOA 01G01112). S.M.C.d. S.L. is supported by the Netherlands Organization of Scientific Research (NWO) (ASPASIA 015.007.037) and the Interuniversity Attraction Poles (PAI) (no. P7/07). P.D.S. is holder of a fundamental clinical research mandate by the FWO. We would like to thank Ferring Company (Aalst, Belgium) for financial support of this study. The authors do not have any competing interests to declare. TRIAL REGISTRATION NUMBER: Not applicable.

Low feasibility of in vitro matured oocytes originating from cumulus complexes found during ovarian tissue preparation at the moment of gender confirmation surgery and during testosterone treatment for fertility preservation in transgender men.

OBJECTIVE: To study the feasibility of in vitro maturation of ovarian tissue oocytes for fertility preservation in transgender men on testosterone treatment. DESIGN: Cross-sectional study SETTING: University hospital PATIENT(S): Eighty-three transgender men enrolled from November 2015 to January 2019 INTERVENTION(S): In vitro maturation of cumulus-oocyte complexes (COCs) harvested at the time of gender confirmation surgery, and fertilization through intracytoplasmic sperm injection. MAIN OUTCOME MEASURE(S): In vitro maturation, fertilization, and blastulation rates; comparison of morphokinetics with vitrified-warmed oocytes; and analysis of the genetic profiles of embryos. SECONDARY OUTCOMES: association between serum hormone levels; COCs' morphologic characteristics, and vitrification rate. RESULT(S): All participants were on testosterone treatment for a median of 83 (64[Quartile 1]; 113.2[Quartile 2]) weeks. A total of 1,903 COCs (mean per participant, 23 ± 15.8) were collected. The in vitro maturation rate was 23.8%, vitrification rate was 21.5%, and survival rate after warming was 72.6% (n = 151). Intracytoplasmic sperm injection was performed in 139 oocytes. The rate of normal fertilized oocytes was 34.5%, and 25 (52.1%) embryos reached day 3. One blastocyst was achieved on day 5. Aberrant cleavage patterns and early embryo arrest were observed in 22 (45.8%) and 44 (91.7%) zygotes, respectively. Compared with vitrified-warmed donor oocytes, a delay was observed in pronuclei disappearance, t2 (time to reach 2 cell stage) timings, and CC1 (the duration of the 1st cell cycle) and SS3 (synchronization of cleavage pattern (calculated as t8-t5) time intervals. A normal genetic pattern was seen in 42% embryos. The proportion of vitrified oocytes was negatively associated with progesterone (odds ratio, 0.76) and positively associated with antimüllerian hormone serum levels (odds ratio, 1.23). The highest vitrification rate was achieved by the morphologic characteristic 344 at day 0 and by 433 at day 2. CONCLUSION(S): Ovarian tissue oocytes matured in vitro show low developmental capacity in transgender men, when collected under testosterone treatment.

Developmental competence of parthenogenetic mouse and human embryos after chemical or electrical activation.

Parthenogenetic reconstruction is one major strategy to create patient-specific stem cells. The aim of this study was to find the best artificial activation protocol for parthenogenetic activation of mouse and human oocytes comparing different methods. In a first set of experiments, in-vivo matured mouse oocytes and human failed-fertilized, in-vitro and in-vivo matured oocytes were artificially activated by a chemical (ionomycin) or electrical stimulus. In a second set of experiments, a combination of activating agents (electrical pulses followed by ionomycin or SrCl(2)) was applied in an aim to improve developmental competence. All embryos were evaluated daily until day 6 after activation. Mouse blastocysts were differentially stained to evaluate blastocyst quality. For mouse oocytes and human failed-fertilized oocytes, blastocyst development was significantly higher after electrical activation (P<0.05). For human in-vitro and in-vivo matured oocytes, blastocyst formation was only obtained after electrical activation of in-vitro matured oocytes. After combining activating agents, no differences in development could be observed. In conclusion, this study revealed that for both mouse and human oocytes development to the blastocyst stage was significantly better after electrical activation compared with chemical activation. Combining activating agents had no further positive effect on developmental potential.

Effect of ionomycin on oocyte activation and embryo development in mouse.

Artificial oocyte activation using the calcium ionophore ionomycin is applied successfully in assisted reproduction but some concern exists on the clinical use. The aims of the present study were to optimize the oocyte activation scheme and to address embryo toxicity in a mouse model. Efficiency of oocyte activation and subsequent development was evaluated and ionomycin was found to be an efficient activator at 10 micromol/l. An improved effect of a second exposure to 5 micromol/l ionomycin on blastocyst development was observed. Toxicity of ionomycin on embryos was then investigated by evaluating pre- and post-implantation development of in-vivo fertilized oocytes following exposure to ionomycin. Blastocyst development, blastocyst cell numbers in trophectoderm and inner cell mass were not different between treated and non-treated zygotes. Also implantation rates and fetal parameters such as length, weight and morphological parameters were similar between the fetuses originating from zygotes treated with ionomycin and non-treated zygotes. Furthermore, healthy offspring originating from ionomycin-treated zygotes was born. In conclusion, no adverse effects of ionomycin on in-vitro or in-vivo mouse embryo development were noticed, giving arguments in favour of the use of ionomycin, although negative long-term effects of this compound cannot be excluded at present.

Inhibition of transforming growth factor ß signaling promotes epiblast formation in mouse embryos.

Early lineage segregation in preimplantation embryos and maintenance of pluripotency in embryonic stem cells (ESCs) are both regulated by specific signaling pathways. Small molecules have been shown to modulate these signaling pathways. We examined the influence of several small molecules and growth factors on second-lineage segregation of the inner cell mass toward hypoblast and epiblast lineage during mouse embryonic preimplantation development. We found that the second-lineage segregation is influenced by activation or inhibition of the transforming growth factor (TGF)ß pathway. Inhibition of the TGFß pathway from the two-cell, four-cell, and morula stages onward up to the blastocyst stage significantly increased the epiblast cell proliferation. The epiblast formed in the embryos in which TGFß signaling was inhibited was fully functional as demonstrated by the potential of these epiblast cells to give rise to pluripotent ESCs. Conversely, activating the TGFß pathway reduced epiblast formation. Inhibition of the glycogen synthase kinase (GSK)3 pathway and activation of bone morphogenetic protein 4 signaling reduced the formation of both epiblast and hypoblast cells. Activation of the protein kinase A pathway and of the Janus kinase/signal transducer and activator of transcription 3 pathway did not influence the second-lineage segregation in mouse embryos. The simultaneous inhibition of three pathways--TGFß, GSK3ß, and the fibroblast growth factor (FGF)/extracellular signal-regulated kinases (Erk)--significantly enhanced the proliferation of epiblast cells than that caused by inhibition of either TGFß pathway alone or by combined inhibition of the GSK3ß and FGF/Erk pathways only.

Application Of Small Molecules Favoring Naïve Pluripotency during Human Embryonic Stem Cell Derivation.

In mice, inhibition of both the fibroblast growth factor (FGF) mitogen-activated protein kinase kinase/extracellular-signal regulated kinase (MEK/Erk) and the Wnt signaling inhibitor glycogen synthase-3ß (GSK3ß) enables the derivation of mouse embryonic stem cells (mESCs) from nonpermissive strains in the presence of leukemia inhibitory factor (LIF). Whereas mESCs are in an uncommitted naïve state, human embryonic stem cells (hESCs) represent a more advanced state, denoted as primed pluripotency. This burdens hESCs with a series of characteristics, which, in contrast to naïve ESCs, makes them not ideal for key applications such as cell-based clinical therapies and human disease modeling. In this study, different small molecule combinations were applied during human ESC derivation. Hereby, we aimed to sustain the naïve pluripotent state, by interfering with various key signaling pathways. First, we tested several combinations on existing, 2i (PD0325901 and CHIR99021)-derived mESCs. All combinations were shown to be equally adequate to sustain the expression of naïve pluripotency markers. Second, these conditions were tested during hESC derivation. Overall, the best results were observed in the presence of medium supplemented with 2i, LIF, and the noncanonical Wnt signaling agonist Wnt5A, alone and combined with epinephrine. In these conditions, outgrowths repeatedly showed an ESC progenitor-like morphology, starting from day 3. Culturing these "progenitor cells" did not result in stable, naïve hESC lines in the current conditions. Although Wnt5A could not promote naïve hESC derivation, we found that it was sustaining the conversion of established hESCs toward a more naïve state. Future work should aim to distinct the effects of the various culture formulations, including our Wnt5A-supplemented medium, reported to promote stable naïve pluripotency in hESCs.

Derivation of human embryonic stem cells using a post-inner cell mass intermediate.

Little is known about the true developmental origin of human embryonic stem cells (hESCs) or the events that initiate their generation. Recently, we have shown that hESCs originate from a post-inner cell mass (ICM) intermediate (PICMI), a unique transient epiblast-like structure that is different from both its ICM progenitor and its subsequent hESC fate. As a closer progenitor of hESCs than the ICM, the PICMI could be used to provide further insight into the human pluripotent state. Here we provide a detailed (7-d) protocol for the culture of the human preimplantation embryos in order to derive the PICMI. Subsequent identification and cryopreservation of the PICMI are described, in addition to hESC derivation. The initial hESC outgrowth is visible within 2-7 d after PICMI plating. By using the protocol provided, we observed PICMI formation in 21.3% of plated blastocysts with good-quality ICMs. Of the PICMIs used for hESC derivation, 80.6% showed hESC outgrowth after further culture.

The combination of inhibitors of FGF/MEK/Erk and GSK3ß signaling increases the number of OCT3/4- and NANOG-positive cells in the human inner cell mass, but does not improve stem cell derivation.

In embryonic stem cell culture, small molecules can be used to alter key signaling pathways to promote self-renewal and inhibit differentiation. In mice, small-molecule inhibition of both the FGF/MEK/Erk and the GSK3ß pathways during preimplantation development suppresses hypoblast formation, and this results in more pluripotent cells of the inner cell mass (ICM). In this study, we evaluated the effects of different small-molecule inhibitors of the FGF/MEK/Erk and GSK3ß pathway on embryo preimplantation development, early lineage segregation, and subsequent embryonic stem cell derivation in the humans. We did not observe any effect on blastocyst formation, but small-molecule inhibition did affect the number of OCT3/4- and NANOG-positive cells in the human ICM. We found that combined inhibition of the FGF/MEK/Erk and GSK3ß pathways by PD0325901 and CHIR99021, respectively, resulted in ICMs containing significantly more OCT3/4-positive cells. Inhibition of FGF/MEK/Erk alone as well as in combination with inhibition of GSK3ß significantly increased the number of NANOG-positive cells in blastocysts possessing good-quality ICMs. Secondly, we verified the influence of this increased pluripotency after 2i culture on the efficiency of stem cell derivation. Similar human embryonic stem cell (hESC) derivation rates were observed after 2i compared to control conditions, resulting in 2 control hESC lines and 1 hESC line from an embryo cultured in 2i conditions. In conclusion, we demonstrated that FGF/MEK/Erk and GSK3ß signaling increases the number of OCT3/4- and NANOG-positive cells in the human ICM, but does not improve stem cell derivation.

Influence of activin A supplementation during human embryonic stem cell derivation on germ cell differentiation potential.

Human embryonic stem cells (hESCs) are more similar to "primed" mouse epiblast stem cells (mEpiSCs). mEpiSCs, which are derived in Activin A, show an increased propensity to form primordial germ cell (PGC)-like cells in response to bone morphogenic protein 4 (BMP4). Hence, we hypothesized that hESCs derived in the presence of Activin A may be more competent in differentiating towards PGC-like cells after supplementation with BMP4 compared to standard hESC lines. We were able to successfully derive two hESC lines in the presence of Activin A, which were pluripotent and showed higher base levels of STELLA and cKIT compared to standard hESC lines derived without Activin A addition. Furthermore, upon differentiation as embryoid bodies in the presence of BMP4, we observed upregulation of VASA at day 7, both at the transcript and protein level compared to standard hESC lines, which appeared to take longer time for PGC specification. Unlike other hESC lines, nuclear pSMAD2/3 presence confirmed that Activin signalling was switched on in Activin A-derived hESC lines. They were also responsive to BMP4 based on nuclear detection of pSMAD1/5/8 and showed endodermal differentiation as a result of GATA-6 expression. Hence, our results provide novel insights into the impact of hESC derivation in the presence of Activin A and its subsequent influence on germ cell differentiation potential in vitro.

Tracking the progression of the human inner cell mass during embryonic stem cell derivation.

The different pluripotent states of mouse embryonic stem cells (ESCs) in vitro have been shown to correspond to stages of mouse embryonic development. For human cells, little is known about the events that precede the generation of ESCs or whether they correlate with in vivo developmental stages. Here we investigate the cellular and molecular changes that occur during the transition from the human inner cell mass (ICM) to ESCs in vitro. We demonstrate that human ESCs originate from a post-ICM intermediate (PICMI), a transient epiblast-like structure that has undergone X-inactivation in female cells and is both necessary and sufficient for ESC derivation. The PICMI is the result of progressive and defined ICM organization in vitro and has a distinct state of cell signaling. The PICMI can be cryopreserved without compromising ESC derivation capacity. As a closer progenitor of ESCs than the ICM, the PICMI provides insight into the pluripotent state of human stem cells.

The influence of early embryo traits on human embryonic stem cell derivation efficiency.

Despite its prognostic value in in vitro fertilization, early embryo morphology is not reported on in the derivation of human embryonic stem cell (hESC) lines. Standard hESC derivation does rely on blastocyst development and its efficiency is highly correlated to inner cell mass (ICM) quality. Poor-quality embryos (PQEs) donated for hESC derivation may have a range of cleavage-stage abnormalities that are known to compromise further development. This study was implemented to determine whether specific PQEs traits influence the efficiency of good-quality ICMs to derive new hESC lines. We found that although the types of PQEs investigated were all able to make blastocysts with good-quality ICMs, the ICMs were unequal in their ability to derive hESCs. Good-quality ICMs from embryos with multiple poor-quality traits were unable to generate hESC lines, in contrast to good-quality ICMs from embryos with a single poor-quality trait. In addition, our data suggest a direct correlation between the number of ICM cells present in the blastocyst and its capacity to derive new hESC lines. This study is the first to demonstrate that ICM quality alone is an incomplete indicator of hESC derivation and that application of in vitro fertilization-based early embryo scoring can help predict hESC derivation efficiency. Experiments aiming to quantify, improve upon, or compare hESC derivation efficiency should thus take into consideration early embryo morphology scoring for the comparison of groups with equal developmental competence.

Double-quality control reveals high-level toxicity in gloves used for operator protection in assisted reproductive technology.

OBJECTIVE: To submit different glove brands to double-quality control tests using mouse embryo assay (MEA) and the human sperm motility assay (HuSMA). Operator protection against infectious body fluid contamination is a safety issue in assisted reproductive technology (ART). When using gloves in the ART laboratory, toxic substances can be transmitted to culture media, even during brief contact. DESIGN: Quality control study of gloves in ART. SETTING: University hospital-based infertility center. ANIMAL(S): Seven- to 8-week-old female B6D2F1 hybrid mice. INTERVENTION(S): We tested two surgical, two cleanroom, and six examination glove brands. Only gloves brands that passed both HuSMA and MEA were submitted to further QC using zona-free and/or cryopreserved MEA. MAIN OUTCOME MEASURE(S): Sperm motility index, two-cell and blastocyst development, blastocyst total cell number. RESULT(S): Quality control by MEA and HuSMA identified two glove brands to be nontoxic. CONCLUSION(S): Our study shows that gloves used in ART can be toxic and should be tested as part of an ongoing quality control program.