KLF17 promotes human naïve pluripotency but is not required for its establishment.

Current knowledge of the transcriptional regulation of human pluripotency is incomplete, with lack of interspecies conservation observed. Single-cell transcriptomics analysis of human embryos previously enabled us to identify transcription factors, including the zinc-finger protein KLF17, that are enriched in the human epiblast and naïve human embryonic stem cells (hESCs). Here, we show that KLF17 is expressed coincident with the known pluripotency-associated factors NANOG and SOX2 across human blastocyst development. We investigate the function of KLF17 using primed and naïve hESCs for gain- and loss-of-function analyses. We find that ectopic expression of KLF17 in primed hESCs is sufficient to induce a naïve-like transcriptome and that KLF17 can drive transgene-mediated resetting to naïve pluripotency. This implies a role for KLF17 in establishing naïve pluripotency. However, CRISPR-Cas9-mediated knockout studies reveal that KLF17 is not required for naïve pluripotency acquisition in vitro. Transcriptome analysis of naïve hESCs identifies subtle effects on metabolism and signalling pathways following KLF17 loss of function, and possible redundancy with other KLF paralogues. Overall, we show that KLF17 is sufficient, but not necessary, for naïve pluripotency under the given in vitro conditions.

Die-off ratio correlates with increased TNF-α:IL-10 ratio and decreased IVF success rates correctable with humira.

BACKGROUND: Human embryos develop at varying rates in culture, with only a fraction of the eggs retrieved developing to 'transfer quality' embryos. We investigated whether the ratios between the number of eggs retrieved or the number of pro-nucleate embryos formed and the number of Day 3 embryos with ≥5 cells [oocyte 'die-off ratios' (DOR)] were correlated with the chance of IVF success, independent of other factors such as embryo grade score and patient's age. We also investigated what factors may be correlated with this ratio. METHODS: 608 IVF fresh cycles in subfertile women were retrospectively evaluated. For each cycle, an oocyte DOR number was calculated as follows: Number of eggs retrieved divided by the number of Day 3 embryos with ≥5 cells. This number was correlated with the subsequent success rates for the index cycles. A 'post-fertilization' or 'embryo' die-off ratio (EDOR; the number of pro-nucleate embryos/the number of day 3 embryos ≥5 cells) was also calculated. RESULTS: The oocyte DOR showed a reverse linear correlation with IVF live birth rate. Live birth rate = (-5.75; DOR) +71.6 (with DOR > 1; P ≤ 0.005; R = -0.87). In addition, the oocyte DOR continued to show an inverse correlation with success rates even when embryo quality and patient's age were held constant. The post-fertilization or EDOR also continued to show a statistically significant negative correlation with live birth rate (R = -0.91; P ≤ 0.01). The preconception TNF-α:IL-10 ratio, an immmunologic marker (drawn 3.3 ± 2.6 months preconception), was more strongly correlated with high oocyte DOR than either age or number of eggs retrieved (P = 0.04, 0.14, 0.72, respectively). When anti-TNF-α therapy (Humira) was given preconception, the oocyte DOR's negative effect on live birth rate was nearly eliminated (correlation coefficient between oocyte DOR and live birth rate: cycles using no Humira, R = -0.90, P ≤ 0.006; cycles using Humira, R = 0.25, P ≤ 0.55). CONCLUSIONS: In subfertile women undergoing IVF, the oocyte DOR may help predict IVF success rates. This factor may offer an additional tool to help improve implantation rate, clinical pregnancy rate, live birth rate, and live birth rate per embryo transferred for an upcoming IVF cycle. Although many mechanisms may contribute to the oocyte DOR's negative effect on IVF success rates, its correlation with elevated preconception TNF-α:IL-10 ratio and correction with Humira suggests a strong immunologic component that may be treatable.

Elevated preconception CD56+ 16+ and/or Th1:Th2 levels predict benefit from IVIG therapy in subfertile women undergoing IVF.

PROBLEM: We sought to answer two questions: First, is there a group of patients who benefit from intravenous immunoglobulin (IVIG) in IVF? Second can this group of patients be identified by preconception blood testing? METHOD OF STUDY: A total of 202 IVF cycles in subfertile women were divided into four groups. Group I: 62 cycles with preconception Th1:Th2 ratio and/or % CD56(+) cell elevation using IVIG; Group II: 27 cycles with similar Th1:Th2 and/or % CD56(+) cell elevation not using IVIG; Group III: 71 cycles with normal Th1:Th2 and/or % CD56(+) cell levels using IVIG; Group IV: 42 cycles with normal Th1:Th2 and % CD56(+) levels not using IVIG. These groups were similar with regard to patient age, diagnosis, and past failure history. RESULTS: The implantation rate (number of gestational sacs per embryo transferred, with an average of two embryos transferred per cycle) was 45% (55/123), 22% (12/54), 54% (75/139), and 48% (40/84) for Groups I-IV, respectively. The clinical pregnancy rate (fetal heart activity per IVF cycle started) was 61% (38/62), 26% (7/27), 69% (49/71), and 71% (30/42), respectively. The live birth rate was 58% (36/62), 22% (6/27), 61% (43/71), and 71% (30/42), respectively, and the live birth per embryo transferred was 40% (49/123), 13% (7/24), 43% (60/139), and 48% (40/84), respectively. There was a significant improvement in implantation, clinical pregnancy, live birth rate and live birth rate per embryo transferred for Group I versus Group II (P = 0.0032, 0.0021, 0.0017, and 0.0002, respectively) and for Group II versus Group IV (P = 0.0021, 0.0002, <0.0001 and <0.0001, respectively). There was no significant difference in success rates between Groups I and III (P = 0.085, 0.23, 0.45, 0.34, respectively) and between Groups III and IV (P = 0.22, 0.48, 0.17, 0.31, respectively). CONCLUSION: In subfertile women with preconception Th1:Th2 and/or % CD56(+) cell elevation, IVF success rates are low without IVIG therapy but significantly improve with IVIG therapy. In patients with normal Th1:Th2 and normal CD56(+) cell levels, IVF success rates were not further improved with IVIG therapy. IVIG may be a useful treatment option for patients with previous IVF failure and preconception Th1:Th2 and/or NK elevation. Preconception immune testing may be a critical tool for determining which patients will benefit from IVIG therapy. Prospective controlled studies (preferably double-blind, stratified, and randomized) are needed for confirmation.

Birth defect rates in women using Adalimumab (Humira(®) ) to treat immunologic-based infertility in IVF patients.

PROBLEM: This study compares the birth defect rate in women using preconception TNF-α inhibitor (Adalimumab) during an in vitro fertilization (IVF) cycle to a similar population of women not using these immunologic therapies. METHOD OF STUDY: One hundred subfertile women aged ≤38years experienced ongoing pregnancies of which 36 resulted in twin pregnancies (136 babies). These successful cycles were divided into two different treatment groups: group I comprised 31 cycles (23 ICSI) using preconception Adalimumab (Humira) with or without pre- or post-conception intravenous immunoglobulin (IVIG) (last dose of Humira given 65.3±41.5days before embryo transfer). Group II comprised 69 cycles (58 ICSI) with no exposure to Humira or IVIG. Group I included all eligible fresh cycles containing at least five 5-celled embryos on day 3. Group II had similar entry criteria, but eligible cycles were randomly selected owing to a larger population size. Patients were later contacted by the clinic for neonatal health information. RESULTS: Delivery outcomes were as follows: group I experienced one case of DiGeorge syndrome (chromosome 22 deletion) that was electively terminated out of 41 babies (10 sets of twins) delivered. Group II experienced one case of neonatal heart defect and another case of Edward's syndrome (Trisomy 18) out of 95 babies (26 sets of twins) delivered. The anomaly rate was 2.44% (1/41) and 2.11% (2/95) for groups I and II, respectively, comparable to the expected birth defect rate for the normal IVF population. CONCLUSION: Preconception TNF-α inhibitor does not appear to increase the birth defect rate in women undergoing IVF. A larger, clinical trial with blinded delivery assessment is needed to confirm these safety conclusions.

Degree of TNF-α/IL-10 cytokine elevation correlates with IVF success rates in women undergoing treatment with Adalimumab (Humira) and IVIG.

PROBLEM: In this retrospective observational study, we investigate whether the degree of preconception cytokine elevation predicts the risk of IVF failure. METHOD OF STUDY: Seventy-six women undergoing fresh IVF/ICSI cycles (≤41 years, good responders with ≥5 embryos on day 3, each with ≥5 cells with a normal endometrium) and preconception tumor necrosis factor (TNF)-α/IL-10 cytokine elevation [PMA/ionomycin stimulated CD3(+) CD8(-) ; TNF-α/IL-10 ratio above 30.6 (normal range 13.2-30.6)] were retrospectively evaluated. This high cytokine population was divided into two subgroups. Group I included 39 women with severe preconception and pre-treatment TNF-α/IL-10 cytokine elevation >39.0 (mean 47.5 ± 7.5 pre-treatment, mean 31.5 ± 9.4 post-treatment) treated with preconception Adalimumab (Humira(®) ) and intravenous immunoglobulin (IVIG). Group II included 37 women with a moderate TNF-α/IL-10 ratio >30.6 and ≤39.0 (mean 35.2 ± 2.2 pre-treatment, mean 28.8 ± 8.5 post-treatment) treated with preconception Adalimumab and IVIG. Groups I and II were comparable in relation to baseline IVF characteristics and patient history. RESULTS: The implantation rate (number of gestational sacs per embryo transfer, with an average of two embryos transferred per cycle) was 43% (36/83) for Group I and 56% (44/78) for Group II. The clinical pregnancy rate (fetal heart activity per IVF cycle started) was 67% (26/39) for Group I and 73% (27/37) for Group II. The delivery rate was 56% (22/39) for Group I and 68% (25/37) for Group II. The live birthrate per embryo transferred was 36% (30/83) for Group I and 45% (35/78) for Group II. Comparing Groups I and II, there was non-significant increase in implantation rate, clinical pregnancy rate, delivery rate, and live birthrate per embryo transferred (P = 0.1, 0.6, 0.4, and 0.3, respectively). However, when the subgroup with the least optimal cytokine conditions (Group I with inadequate cytokine suppression) was compared to the subgroup with the most optimal cytokine conditions (Group II with adequate cytokine suppression), the increase in implantation rate reached statistical significance [41% (19/46) to 64% (29/45); P = 0.04]. The reduction in TNF-α/IL-10 ratio following immunotherapy was highly significant (P < 0.0001). CONCLUSION: The degree of preconception TNF/IL-10 elevation may correlate with an increased risk of IVF failure. Elevated TNF-α/IL-10 ratios can be corrected with therapy. It may be possible to improve IVF success rates by modulating high cytokine levels. Although our pilot database is small, the trends in the data are consistent and compelling. Larger studies are needed for confirmation.

Cell bio-imaging reveals co-expression of HLA-G and HLA-E in human preimplantation embryos.

The non-classical major histocompatibility complex (MHC) class Ib antigens, termed HLA-G and HLA-E, have been associated with fetal maternal tolerance. The role of HLA-G in the preimplantation embryo remains unclear although immunoprotection, adhesion and cell signalling mechanisms have been suggested. Unlike HLA-G, HLA-E protein expression has not been previously studied in preimplantation embryos. Embryos and model trophoblast cell lines JEG-3 and BeWo were labelled with the HLA-G- and HLA-E-specific monoclonal antibodies MEMG9 and MEME07. Flow cytometry, confocal microscopy and single particle fluorescence imaging techniques were employed to investigate the spatial and temporal expression of these receptors. Lipid raft analysis and adhesion assays were performed to investigate the role of these receptors in cell membrane domains and in promoting adhesion by cell-to-cell contact. HLA-E and HLA-G were co-localized in the trophectoderm of day 6 blastocysts. Analysis on trophoblast cell lines revealed that 37% of HLA-G and 41% of HLA-E receptors were co-localized as tetramers or higher order homodimer clusters. HLA-G receptors did not appear to play a role in either cell adhesion or immunoreceptor signalling via lipid raft platforms on the cell membrane. A possible role of HLA-G and HLA-E in implantation via immunoregulation or modulation of uterine maternal leukocytes is discussed.

Treatment with adalimumab (Humira) and intravenous immunoglobulin improves pregnancy rates in women undergoing IVF.

PROBLEM: The purpose of this study was to investigate whether treatment with TNF-alpha inhibitors and/or intravenous immunoglobulin (IVIG) increases in vitro fertilization (IVF) success rates among young (<38 years) women with infertility and T helper 1/T helper 2 cytokine elevation. METHOD OF STUDY: Seventy-five sub-fertile women with Th1/Th2 cytokine elevation were divided into four groups: Group I: Forty-one patients using both IVIG and Adalimumab (Humira), Group II: Twenty-three patients using IVIG, Group III: Six patients using Humira, and Group IV: Five patients using no IVIG or Humira. RESULTS: The implantation rate (number of gestational sacs per embryo transferred, with an average of two embryos transferred by cycle) was 59% (50/85), 47% (21/45), 31% (4/13) and 0% (0/9) for groups I, II, III and IV respectively. The clinical pregnancy rate (fetal heart activity per IVF cycle started) was 80% (33/41), 57% (13/23), 50% (3/6) and 0% (0/5) and the live birth rate was 73% (30/41), 52% (12/23), 50% (3/6) and 0% (0/5) respectively. There was a significant improvement in implantation, clinical pregnancy and live birth rates for group I versus group IV (P = 0.0007, 0.0009, and 0.003, respectively) and for group II versus group IV (P = 0.009, 0.04 and 0.05, respectively). CONCLUSION: The use of a TNF-alpha inhibitor and IVIG significantly improves IVF outcome in young infertile women with Th1/Th2 cytokine elevation.

Analysis of HLA-G in maternal plasma, follicular fluid, and preimplantation embryos reveal an asymmetric pattern of expression.

Soluble HLA-G (sHLA-G) secretion by human preimplantation embryos in culture has been associated with successful embryo development, and therefore has potential to serve as a noninvasive marker of embryo viability. We have examined the spatial and temporal expression of HLA-G in embryos of varying developmental competence and the role of maternal factors in human embryonic HLA-G expression. Embryos that reached blastocyst stage on day 5 showed a higher frequency of sHLA-G secretion than those at morula or arrested stages (p < 0.05). There was no significant difference in sHLA-G secretion between normal embryos and those diagnosed as chromosomally abnormal by preimplantation genetic diagnosis. HLA-G detected in maternal plasma and follicular fluid did not appear to correlate with HLA-G expressed in the embryo or embryo supernatants. Confocal microscopy analysis indicated that HLA-G protein expression in embryos was not homogeneous; mostly, it was confined to blastocysts localized on trophectoderm and trophectoderm projections. Single-particle fluorescent imaging analysis of HLA-G on the cell surface of JEG-3 cells showed that HLA-G particles were mostly monomeric, but dimeric and higher order oligomers were also observed. These results suggest that HLA-G play an important role in preimplantation embryo development. However, the observed expression of HLA-G in arrested and chromosomally abnormal embryos indicates that HLA-G testing should be used with caution and in conjunction with conventional methods of embryo screening and selection.

Towards better quality research in recurrent implantation failure: standardizing its definition is the first step.

Recurrent implantation failure is a frustrating condition for clinicians and patients alike. The number of potential therapies offered to patients in order to overcome this problem is increasing, and more research is needed to establish which of those treatment options is truly beneficial. Improved understanding of their value is more likely if the same definition of recurrent implantation failure is used across future studies. In this article, the inconsistency present in current literature is examined and the case is argued for a standardized definition for the condition.

Influence of maternal age on the outcome of PGD for aneuploidy screening in patients with recurrent implantation failure.

This study assessed the influence of maternal age on the outcome of aneuploidy screening (AS) cycles for recurrent implantation failure (RIF). One hundred and sixteen couples with a history of RIF underwent 130 cycles of AS. Group A included 78 patients aged < or = 40 years (range 25-40 years) who underwent 86 cycles, while group B included 38 couples aged > or = 41 (range 41-47) who underwent 44 cycles. Fluorescence in-situ hybridization (FISH) analysis of the first and second polar bodies using probes specific for chromosomes 13, 16, 18, 21 and 22 was conducted. Euploid oocytes that cleaved were subsequently tested using the same probes on a single blastomere obtained from day 3 embryos. Chromosomally normal embryos were replaced on day 5 of culture. There was no significant difference between the two groups in the mean numbers of oocytes fertilized normally and oocytes (7.5 +/- 3.2 versus 7.2 +/- 3.6) and embryos tested (4.1 +/- 3 versus 3.4 +/-3). However, the younger age group had a significantly higher proportion of euploid oocytes/embryos, cycles reaching embryo transfer, pregnancy (43 versus 25%), clinical pregnancy (36.1 versus 16.6%) and ongoing delivery (32 versus 12.5%) rates per transfer. Preimplantation genetic diagnosis with AS for recurrent IVF implantation failure using FISH probes is therefore associated with improved outcome in women under 41 years, but has a high cancellation rate and low cycle outcome in older women.