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PurposeUsing a comprehensive flow cytometric panel, simultaneously obtained mid-luteal immunophenotypes from peripheral blood and endometrium were compared and values correlated. Is a peripheral blood evaluation of reproductive immunophenotype status meritorious relative to local endometrial evaluation to directly assess the peri-implantation environment?MethodsFifty-five patients had a mid-luteal biopsy to assess the local endometrial immunophenotype, while simultaneously providing a peripheral blood sample for analysis. Both samples were immediately assessed using a comprehensive multi-parameter panel, and lymphocyte subpopulations were described and compared.ResultsDistinct lymphocyte proportions and percentage differences were noted across the two compartments, confirming the hypothesis that they are distinct environments. The ratio of CD4 + to CD8 + T cells were reversed between the two compartments, as were Th1 and Th2-type CD4 + T cell ratios. Despite these differences, some direct relationships were noted. Positive Pearson correlations were found between the levels of CD57 + expressing natural killer cells, CD3 + NK-T cells and CD4 + Th1 cells in both compartments.ConclusionsFlow cytometric evaluation provides a rapid and objective analysis of lymphocyte subpopulations. Endometrial biopsies have become the gold standard technique to assess the uterine immunophenotype in adverse reproductive outcome, but there may still a place for peripheral blood evaluation in this context. The findings demonstrate significant variations in cellular proportions across the two regions, but some positive correlations are present. Immunological assessment of these specific peripheral blood lymphocyte subtypes may provide insight into patients with potential alterations of the uterine immune environment, without the risks and inconveniences associated with an invasive procedure.

Purpose Endometrial platelet-rich plasma (PRP) is proposed to treat thin endometrium and implantation failure. Different techniques are described, and nomenclature is often confusing, with infusion or injection being used interchangeably, not discriminating between methods. Sub-endometrial injection, by hysteroscopic or ultrasound guidance, differs from intracavity infusion, but whether this method is superior has not been subject to review. Methods A systematic review and meta-analysis was performed to compare sub-endometrial injection against placebo or intra-cavity infusion in a defined assisted reproduction population, with subgroup analysis to assess for any differences between USS-guided or Hysteroscopic Techniques. Primary outcomes were clinical pregnancy (CPR), live birth (LBR), and miscarriage rates (MR). Results Significant increases in CPR (OR = 5.14, p  < 0.001) and LBR (OR = 4.60, p  < 0.001) were seen in appropriately selected patients with sub-endometrial PRP injection compared to placebo, with a reduction in MR (OR = 0.60, p  = 0.036). The benefit of injection compared to infusion appears highest for CPR in those with a resistant thin endometrium ( p  = 0.03). Conclusions Sub-endometrial administration may improve the efficacy of PRP in selected patients. More work is needed to identify the optimal candidates, and given the additional administration challenges, whether injection should be a first-line intervention, or considered after unsuccessful infusion, remains open to debate, with more studies needed to confirm this.

Obese or overweight patients considering IVF are generally counselled to reduce weight closer to target BMI (i.e., < 30 kg/m 2 ) by interventions entailing dietary change with a structured exercise program. There is little disagreement that supervised weight loss can improve reproductive outcome when successful, although there are refractory cases where weight goals are unmet. Because low-grade chronic inflammation and altered immune function are characteristic of obesity and antagonize implantation, any pre-IVF weight loss facilitated by semaglutide (SG) would be helpful. However, no preclinical data have considered the ovarian implications of SG. Several formulations of SG are now available to assist in chronic weight management, treatment of type-2 diabetes, or both. SG is 31-amino acid lipopeptide with action at the glucagon-like peptide-1 (GLP-1) receptor, which augments insulin secretion while lowering hepatic glucagon output. SG thus enters a multiorgan network where insulin, AMP-activated protein kinase (AMPK), insulin-like growth factor-1 (IGF-1), mammalian target of rapamycin (mTOR), and sirtuin pathways manage ambient nutritional conditions. As GLP-1 directly influences insulin release and curtails satiety, SG adjusts many biochemical cascades where potential interference with oocyte development or embryo/endometrial crosstalk require clarification. Particularly if used outside manufacturer’s guidance (i.e., for aesthetic or personal reasons), SG could bring unwelcome challenges to fertility clinics where obesity and dyslipidemia are merely exchanged for the new problems of starvation and sarcopenia. Here we examine known GLP-1 actions where energy balance, ovarian aging, and oocyte competence converge; off label SG use should be avoided until its signaling effects throughout the reproductive axis are more carefully studied.

PurposeThe uterine immunophenotype is relatively poorly understood, with most studies reporting proportions/percentages. A novel technique to calculate local endometrial lymphocyte concentrations is described, and used to compare results between aetiological subgroups such as repeated implantation failure (RIF) and recurrent pregnancy loss (RPL) with male-factor controls.Methods455 patients had an endometrial biopsy performed. Background history on initial presentation was used to subdivide the population into RIF (n = 149), RPL (n = 121), primary (n = 76) and secondary infertility (n = 80). A control group was identified comprising male factor infertility aetiology with all female investigations normal (n = 29). Endometrial Tissue was assessed using a comprehensive multi-parameter panel. Lymphocyte subpopulations were calculated using flowcount flurospheres and a mathematical correction applied to determine concentrations per milligram of tissue, based on original biopsy weight and volumetric dilutions.ResultsThe flow cytometry technique was successful in determining population centiles for concentrations of endometrial lymphocyte subsets. Distinct differences were noted across the patient groups. Th2 concentrations were significantly higher in the controls (p = 0.0002). All RPL/infertile populations had increased concentrations of peripheral type NK’s (p = 0.016) and B cells (p = 0.045). Relative to male factor controls, CD4+ and CD8+ T lymphocyte populations were increased in RPL patients, and reduced in those with a history of RIF. Th1 concentrations were elevated in the adverse outcome groups (p = 0.032). Concentration centiles alone do not appear to accurately predict outcome with subsequent treatment.ConclusionsEndometrial biopsy analysis by flow cytometry can provide detailed analysis of constituent lymphocyte subsets by concentration as well as proportion. This novel approach provides additional independent data to further assess the significance of endometrial changes in the setting of reproductive failure.

PurposeUsing a comprehensive flow cytometric panel, do endometrial immune profiles in adverse reproductive outcomes such as repeat implantation failure (RIF) and repeat pregnancy loss (RPL) differ from each other and male-factor controls?MethodsSix-hundred and twelve patients had an endometrial biopsy to assess the immunophenotype. History on presentation was used to subdivide the population into recurrent implantation failure (RIF) [n = 178], recurrent pregnancy loss (RPL) [n = 155], primary infertility [n = 130] and secondary infertility [n = 114]. A control group was utilised for comparative purposes [n = 35] and lymphocyte subpopulations were described.ResultsDistinct lymphocyte percentage differences were noted across the populations. Relative to controls and RPL, patients with a history of RIF had significantly raised uterine NKs (53.2 vs 45.2 & 42.9%, p < 0.0001). All sub-fertile populations had increased percentage peripheral type NKs (p = 0.001), and exhibited increased CD69+ activation (p = 0.005), higher levels of B cells (p < 0.001), elevated CD4:CD8 ratio (p < 0.0001), lower T-regs (p = 0.034) and a higher proportion of Th1+ CD4s (p = 0.001). Patient aetiology confers some distinct findings, RPL; pNK, Bcells and CD4 elevated; RIF; uNK and CD56 raised while CD-8 and NK-T lowered.ConclusionsFlow cytometric endometrial evaluation has the ability to provide a rapid and objective analysis of lymphocyte subpopulations. The findings show significant variations in cellular proportions of immune cells across the patient categories relative to control tissue. The cell types involved suggest that a potential differential pro-inflammatory bias may exist in patients with a history of adverse reproductive outcomes. Immunological assessment in appropriate populations may provide insight into the underlying aetiology of some cases of reproductive failure.

Genetic features are currently unknown in myelinated retinal nerve fibers (MRNF). For a 20-year-old asymptomatic female with unilateral MRNF, we performed whole genome sequencing (WGS) by standard workflow protocol to produce contiguous long-read sequences with Illumina DNA PCR-Free Prep. After tagmentation, libraries were sequenced on separate runs via NovaSeq 6000 platform at 2 x 150bp read length. Gene variants included rs2248799, rs2672589, rs7555070, rs247616_T and rs2043085_C all associated with an increased macular degeneration risk, and seven novel variants of uncertain significance. For optic disc enlargement, variants rs9988687_A, rs11079419_T, rs6787363 and rs10862708_A suggested an increased risk for this condition. In contrast, modeling revealed retinal detachment risk was reduced by variants identified at rs9651980_T, rs4373767_T, and rs7940691_T which were among five other previously unreported variants. WGS data placed proband at the 66th and 64th percentiles for disc anomaly and retinal detachment risk, respectively. Additionally, risk determined from 16 loci associated with age-related macular degeneration found the patient to be at the 18th percentile for this diagnosis (i.e., below average genetic predisposition). Fundoscopic findings showed mean RNFL thickness was lower with MRNF (77 OS vs. 96μm OD) and RNFL symmetry was impaired (43 %) but stable between 2020 and 2023. Rim area and cup volume were also substantially different (2.33 OS vs. 1.34mm2 OD, and 0.001 OS vs. 0.151mm3 OD, respectively). As the first known evaluation of MRNF via WGS, these data reveal a mixed picture with variants associated with different risks for potentially related ocular pathologies. In addition, we identify multiple new variants of unknown significance. Factors affecting gene expression in MRNF require further study.

BackgroundA complete reproductive immunophenotype is poorly described, with most focus on peripheral blood natural killer cells rather than uterine populations. There is debate regarding normal endometrial levels, with no consensus, and much controversy on correlation with implantation/miscarriage.AimsDevelopment and validation of a rapid endometrial assessment flow cytometry (FCM) technique, allowing determination of local lymphocyte subset ranges, comparison to peripheral blood, and patient subgroup analysis.MethodsProspective pilot, assessing patients with prior implantation, failure offered endometrial biopsy before subsequent ART cycle, functioning as therapeutic scratch. HRT regime administered to standardise environment, and progesterone-primed mid-luteal biopsy (five completed days progestogen, P+5) analysed using comprehensive flow panel to identify lymphocyte subsets.ResultsTwo hundred patients were recruited in a tertiary university-affiliated ART centre. FCM identified differing lymphocyte ranges between peripheral blood and biopsy. Uterine/decidual natural killer cells are the dominant endometrial subtype. Patients with repeated implantation failure had higher uNK levels (52.4 vs 43.7%, p = 0.01). Conversely, B lymphocytes (0.87 vs 0.72%, p = 0.032), pNK (1.21 vs 0.8%, p = 0.041), and NK-T (2.68 vs 2.26, p = 0.031) cells were higher in recurrent pregnancy loss.ConclusionFCM is widely used to assess cellular populations, but not typically employed for endometrial evaluation. FCM provides a rapid, detailed, and quantitative analysis and reduces inter-observer subjectivity bias. Detailed understanding of the normal endometrial immunophenotype, and associated deviations, may provide insight into the aetiology of infertile patients labelled “unexplained”. Failure despite transfer of high grade, or proven euploid blastocysts, is a difficult problem, and endometrial profiling may help identify research areas to determine potential future therapeutic interventions for this difficult to treat population.

No major breakthroughs have entered mainstream clinical fertility practice since egg donation and intracytoplasmic sperm injection decades ago, and oocyte deficits secondary to advanced age continue as the main manifestation of diminished ovarian reserve. In the meantime, several unproven IVF ‘accessories’ have emerged including so-called ovarian rejuvenation which entails placing fresh autologous platelet-rich plasma (PRP) directly into ovarian tissue. Among cellular responses attributed to this intervention are reduced oxidative stress, slowed apoptosis and improved metabolism. Besides having an impact on the existing follicle pool, platelet growth factors might also facilitate de novo oocyte recruitment by specified gene upregulation targeting uncommitted ovarian stem cells. Given that disordered activity at the mechanistic target of rapamycin (mTOR) has been shown to exacerbate or accelerate ovarian aging, PRP-discharged plasma cytokines combined with mTOR suppression by pulsed/cyclic rapamycin represents a novel fusion technique to enhance ovarian function. While beneficial effects have already been observed experimentally in oocytes and embryos with mTOR inhibition alone, this proposal is the first to discuss intraovarian platelet cytokines followed by low-dose, phased rapamycin. For refractory cases, this investigational, tailored approach could amplify or sustain ovarian capacity sufficient to permit retrieval of competent oocytes via distinct but complementary pathways—thus reducing dependency on oocyte donation.

Purpose The purpose of this study was to evaluate the serum estradiol (E2) per oocyte ratio (EOR) as a function of selected embryology events and reproductive outcomes with IVF. Methods This retrospective analysis included all IVF cycles where oocyte collection and fresh transfer occurred between January 2001 and November 2012 at a single institution. Patients were divided by three age groups (<35, 35–39, and ≥40 years) and further stratified into nine groups based on EOR (measured in pmol/L/oocyte). Terminal serum E2 (pmol/mL) was recorded on day of hCG trigger administration, and fertilization rate, cleavage rate, number of good quality embryos, and reproductive outcomes were recorded for each IVF cycle. Results During the study interval, 9109 oocyte retrievals were performed for 5499 IVF patients (mean = 1.7 cycles/patient). A total of 63.4 % of transfers were performed on day 3 ( n  = 4926), while 36.6 % were carried out on day 5 ( n  = 2843). Clinical pregnancy rates were highest in patients with EOR of 250–750 and declined as this ratio increased, independent of patient age. While the odds ratio (OR) for clinical pregnancy where EOR = 250–750 vs. EOR > 1500 was 3.4 ( p  < 0.001; 95 % CI 2.67–4.34), no statistically significant correlation was seen in fertilization, cleavage rates or number of good quality embryos as a function of EOR. Conclusions Predicting reproductive outcomes with IVF has great utility both for patients and providers. The former have the opportunity to build realistic expectations, and the latter are better able to counsel according to measured clinical parameters. A better understanding of follicular dynamics and ovarian response to gonadotropin stimulation could optimize IVF treatments going forward.

Background The origins of adverse reproductive outcome can be multifactorial, but the contribution of the maternal immune system is considered debatable. Elevated intracellular cytokine ratios have been proposed, although not universally supported, as a marker for immunological dysfunction in implantation and early pregnancy. Poor patient selection or inadequate treatment or testing may be confounding factors. Specific immunomodulation, in carefully selected sub-populations of ART patients with poor reproductive history, despite transfer of good quality blastocysts, may potentially improve clinical outcomes. Methods Intracellular cytokine ratios (CKR) were prospectively assessed in 337 patients presenting with a history of implantation failure and/or pregnancy loss, prior to further treatment, and were found to be elevated in 150 (44.5%). Of this group, 134 agreed to initiate a standardised immunotherapy regime (nutraceuticals, prednisolone & intralipids) to evaluate the efficacy of this proposed therapy. Of the intervention population, a small cohort ( n  = 70) delayed commencing ART for ~ 10 weeks to assess if extended pre-treatment nutraceutical supplementation could normalise CKRs prior to starting ART, and if this conferred additional benefit. Results Baseline assessment in the intervention population ( n  = 134) identified 160 miscarriages from 180 total pregnancies (89% miscarriage rate, MR), conceived both spontaneously and by assisted reproduction. Post-treatment analysis of subsequent ART cycles revealed a significant improvement in both implantation (OR 3.0, 2.0–4.5) and miscarriage rates (41/97, 42.2% MR, P  < 0.001). Interestingly, pre-treatment normalisation of CKRs appeared to impart marginal extra benefit prior to subsequent fertility treatment with immunotherapy. Conclusions Following immunomodulation, significant improvements in both implantation rate and miscarriage rate were seen in this poor prognosis population. This suggests a possible role for both detailed immuno-evaluation of patients with poor reproductive history with good embryo quality, and application of personalised immunotherapy regimes alongside ART in selected cases. Future randomised controlled trials are needed to definitively evaluate this potentially promising therapeutic approach.