Correlation of peripheral blood and endometrial immunophenotyping in ART: is peripheral blood sampling useful?

PURPOSE: Using 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? METHODS: Fifty-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. RESULTS: Distinct 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. CONCLUSIONS: Flow 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.

Potential utility of a non-invasive menstrual blood immunophenotype analysis in reproductive medicine.

Purpose Can a comprehensive flow cytometry panel be used to assess immunophenotype profiles in menstrual blood of patients experiencing reproductive failure and age matched controls of proven fertility? Methods 58 recurrent pregnancy loss and repeated implantation failure patients, along with 15 age matched controls of proven fertility, had menstrual blood samples obtained within the first 24 hours of the onset of menstruation to non-invasively assess the local immunophenotype. Using a comprehensive multi-parameter flow panel the lymphocyte sub-populations were described and compared. Results Relative to well established peripheral blood immunophenotyping values, distinct lymphocyte population differences were noted between the subgroups. The ratios of CD4+ and CD8+ T-cells were inverted relative to peripheral blood and uterine NK cells represented by CD56bright were distinctly visualised, emphasising the distinction of menstrual and peripheral blood. Relative to controls there were marked increases in CD3+ve T-cells (p=0.009), CD4:CD8 ratio (p=0.004), CD19 B-cells (p=0.026) and CD56dim NK's (p=0.002) in the reproductive failure cases. Conclusions Flow cytometric evaluation can provide a rapid and objective analysis of lymphocyte subpopulations in many forms of tissue and fluid. The findings show significant variations in cellular composition of immune cells indicating a distinct compartment, with differences between cases and controls. Immunological assessment of the menstrual blood immunophenotype, in clinically appropriate patients, may provide insight into the aetiology of adverse reproductive outcome, without the risks and inconveniences associated with a more invasive endometrial biopsy.

Intralipid therapy and adverse reproductive outcome: is there any evidence?

Controversy exists regarding the benefits of intravenous intralipid therapy in patients with a poor reproductive history. It is frequently reported that there is no evidence to support the effectiveness, utility or safety for this treatment. While individual studies may be perceived as weak, a systematic review and meta-analysis were performed to determine if there is any advantage to patients. PubMed, Embase and Scopus searches were performed with the target populations being either recurrent pregnancy loss (RPL), or recurrent implantation failure (RIF) undergoing assisted reproductive technology (ART) and receiving intralipid infusions. These cohorts were compared with either placebo, no intervention or alternative treatments. The most relevant outcome measures were considered to be clinical pregnancy rate (CPR), live birth rate (LBR), implantation rate (IR) and miscarriage rate (MR). Twelve studies encompassing 2676 participants met the criteria for selection and were included and reviewed. Treatment of the target population with intralipid led to an improvement in IR (Odds Ratio (OR): 2.97, 2.05-4.29), pregnancy rate (OR: 1.64, 1.31-2.04), and LBR (OR: 2.36, 1.75-3.17), with a reduction in MR (OR: 0.2, 0.14-0.30). Although intravenous intralipid is not recommended as a routine treatment for recurrent miscarriage or implantation failure, there is enough data to suggest consideration in selected patients where routine testing is unremarkable, standard treatments have failed and immunological risk factors are present. The presence of abnormal uterine natural killer (uNK) cells needs more study as a target marker to determine those who could benefit. LAY SUMMARY: There is controversy regarding the benefits and efficacy of intravenous intralipid therapy in patients with a poor reproductive history. It is frequently reported that there is no credible evidence to support their use. A situation we frequently face as medical professionals is patients asking us to consider immune therapy (such as intralipid) for reproductive failure where good quality embryos have been used. Intralipid infusions have been reported to improve pregnancy rates with IVF, and reduce the miscarriage risk in selected patient groups, but study results are not universally accepted. We have performed a detailed review and analysis of the literature to determine if there is any benefit to this immune treatment in specific patient groups. Our paper identified and analyzed 12 studies, finding that treatment with intravenous intralipid leads to an improvement in implantation, pregnancy and live birth rates, with a decrease in miscarriage rate. This study shows that there is evidence to suggest consideration of intralipid in certain patients where standard treatments have failed.

Cytometric assessment of uterine receptivity via epithelial ß3 integrin expression.

RESEARCH QUESTION: Many techniques now exist to assess the receptivity status of the endometrium. Can a simple low-cost flow cytometric technique be used to rapidly assess uterine receptivity via a luteal phase endometrial biopsy? DESIGN: Epithelial ß3 integrin (EB3) evaluation was undertaken in 300 women presenting with repeated implantation failure or recurrent pregnancy loss who subsequently underwent 710 assisted reproductive technology (ART) cycles. Endometrial tissue was mechanically dissociated and evaluated using specific antibodies to capture integrin expression. Autologous and donor oocyte embryo transfers were evaluated. A 'High', 'Borderline' and 'Low' grading system was developed based on the pattern and percentage expression of EB3 relative to the total endometrial epithelium. Clinical outcomes of the resulting embryo transfers (n = 559) were analysed according to EB3 grading. RESULTS: Based on 180 completed transfers, the clinical pregnancy rate (CPR) per embryo transferred in the donor egg cycles was 41.7%. EB3 results from women with a 'High' grading showed a superior CPR (54.0%) compared with 'Low' (22.2% CPR) or 'Borderline' (37.4%) cases (P = 0.024). Similarly, following 379 autologous oocyte transfers, the CPR was 36.1% overall, with major variations between the 'High' (43.8%), 'Low' (17.5%) and 'Borderline' (34.8%) groups (P = 0.0008). Implantation rates showed similar significant trends in the 'High' versus 'Low' groups of 40.4% versus 16% (P = 0.048) in donor oocyte transfers, and 30.8% versus 16.1% (P = 0.025) in autologous oocyte transfers. CONCLUSIONS: The distribution patterns and percentage expression of EB3 assessed by a flow cytometry grading system shows a significant relationship to implantation rate and CPR success in ART cycles and may thus represent a useful additional tool for the assessment of uterine receptivity.

Endometrial lymphocyte concentrations in adverse reproductive outcome populations.

PURPOSE: The 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. METHODS: 455 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. RESULTS: The 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. CONCLUSIONS: Endometrial 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.

Moving from peripheral blood to local uterine immunophenotype analysis in patients with poor reproductive history: pilot study of a novel technique.

BACKGROUND: A 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. AIMS: Development 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. METHODS: Prospective 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. RESULTS: Two 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. CONCLUSION: FCM 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.

Detailed endometrial immune assessment of both normal and adverse reproductive outcome populations.

PURPOSE: Using 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? METHODS: Six-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. RESULTS: Distinct 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. CONCLUSIONS: Flow 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.

ART implantation failure and miscarriage in patients with elevated intracellular cytokine ratios: response to immune support therapy.

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.

Anti-oxidant mediated normalisation of raised intracellular cytokines in patients with reproductive failure.

BACKGROUND: Raised intracellular cytokine ratios (CKR) are proposed as a significant risk factor for adverse reproductive outcome. An elevated cytokine ratio, such as between TNFa and/or IFNg to IL-10 is associated with recurrent miscarriage (RM). The use of pharmacological immunomodulators such as TNFα inhibitors in these patients is controversial and not generally recommended due to a lack of conclusive data supporting their use. We evaluated whether the use of anti-oxidants/dietary supplements as an alternative could positively influence CKR's in ART patients. METHODS: A prospective non-placebo control trial of antioxidant treatment for abnormal peripheral inflammatory cytokine ratios was performed. CKRs were assessed using flow cytometry in stimulated versus unstimulated whole blood samples in 337 IVF patients presenting with a previous history of poor outcome (RM or implantation failure). CKR's were found to be elevated in 150/337. 70/150 patients in this elevated group agreed to a 10 week regime of Omega 3, vitamin D3, and B complex, followed by retesting to evaluate effect. RESULTS: Mean cytokine ratios significantly improved between tests. Pre-treatment TNFa:IL-10 ratio improved from 71.6 to 21.0 (p < 0.0001) and IFNg:IL-10 ratio dropped from 24.5 to 12.5 (p < 0.0001). The improved ratios were achieved primarily by an increase in IL-10 expression (P = 0.0007), but also by a moderate decrease in stimulated TNFa expression (p = 0.008). Mean IFNg expression was unchanged (p = 0.42). On an individual basis CKR levels were normalised in 43 patients, improved in 12 and remained unchanged in 15. No significant differences in improvement were found between RM and IF subgroups. CONCLUSIONS: Intracellular cytokine expression levels and ratios were modifiable by the supplement regime employed. Elevated cytokine ratios have been linked with adverse reproductive outcomes, and proposed treatments have included biological immunomodulators which antagonise TNFa, but come with significant associated cost implications and more importantly, cytotoxic side-effects. A dietary regime is more patient friendly and lower risk, while still achieving a similar effect in many patients.

The impact of FMR1 gene mutations on human reproduction and development: a systematic review.

PURPOSE: This is a comprehensive review of the literature in this field attempting to put the FMR1 gene and its evaluation into context, both in general and for the reproductive health audience. METHODS: Online database search of publications with systematic review of all papers relevant to ovarian reserve and assisted reproduction was done. RESULTS: Relevant papers were identified and assessed, and an attempt was made to understand, rationalize and explain the divergent views in this field of study. Seminal and original illustrations were employed. CONCLUSIONS: FMR1 is a highly conserved gene whose interpretation and effect on outcomes remains controversial in the reproductive health setting. Recent re-evaluations of the commonly accepted normal range have yielded interesting tools for possibly explaining unexpected outcomes in assisted reproduction. Fragile X investigations should perhaps become more routinely assessed in the reproductive health setting, particularly following a failed treatment cycle where oocyte quality is thought to be a contributing factor, or in the presence of a surprise finding of diminished ovarian reserve in a young patient.

Ovarian dysgenesis associated with an unbalanced X;6 translocation: first characterisation of reproductive anatomy and cytogenetic evaluation in partial trisomy 6 with breakpoints at Xq22 and 6p23.

The aim of this study was to describe the clinical and laboratory findings associated with a previously unreported unbalanced X;6 translocation. Physical examination, reproductive history and cytogenetic techniques were used to characterise a novel chromosomal anomaly associated with gonadal dysgenesis. A healthy non-dysmorphic 23 year-old phenotypic female with primary amenorrhea and infertility presented for reproductive endocrinology evaluation. No discrete ovarian tissue was identified on transvaginal ultrasound, although the uterus appeared essentially normal. BMI was 19 kg/m2. Serum FSH and oestradiol were 111 mIU/ml and 15 pmol/l, respectively. TSH, prolactin and all infectious serologies were all normal. The karyotype of 46,X,der(X)t(X;6)(q22;p23) was determined following cytogenetic analysis of peripheral blood lymphocytes via fluorescence in situ hybridisation (FISH) with whole chromosome paint for chromosome 6, and a separate FISH analysis using a 6p subtelomeric probe. The patient was continued on hormone replacement therapy and underwent genetic counselling; the patient subsequently enrolled as a recipient in an anonymous donor oocyte IVF treatment. Translocations involving autosomes and chromosome X are rare. While female carriers of balanced X;autosome translocations are generally phenotypically normal, the impact of unbalanced X;autosome translocations can be severe. This is the first known report of an unbalanced translocation involving X;6. This abnormality was associated with ovarian dysgenesis, but an otherwise normal female phenotype. From this investigation, the observed developmental impact of the unbalanced translocation with breakpoints at Xq22 and 6p23 appears to be limited to ovarian failure.

Bivariate analysis of basal serum anti-Müllerian hormone measurements and human blastocyst development after IVF.

BACKGROUND: To report on relationships among baseline serum anti-Müllerian hormone (AMH) measurements, blastocyst development and other selected embryology parameters observed in non-donor oocyte IVF cycles. METHODS: Pre-treatment AMH was measured in patients undergoing IVF (n = 79) and retrospectively correlated to in vitro embryo development noted during culture. RESULTS: Mean (+/- SD) age for study patients in this study group was 36.3 ± 4.0 (range = 28-45) yrs, and mean (+/- SD) terminal serum estradiol during IVF was 5929 +/- 4056 pmol/l. A moderate positive correlation (0.49; 95% CI 0.31 to 0.65) was noted between basal serum AMH and number of MII oocytes retrieved. Similarly, a moderate positive correlation (0.44) was observed between serum AMH and number of early cleavage-stage embryos (95% CI 0.24 to 0.61), suggesting a relationship between serum AMH and embryo development in IVF. Of note, serum AMH levels at baseline were significantly different for patients who did and did not undergo blastocyst transfer (15.6 vs. 10.9 pmol/l; p = 0.029). CONCLUSIONS: While serum AMH has found increasing application as a predictor of ovarian reserve for patients prior to IVF, its roles to estimate in vitro embryo morphology and potential to advance to blastocyst stage have not been extensively investigated. These data suggest that baseline serum AMH determinations can help forecast blastocyst developmental during IVF. Serum AMH measured before treatment may assist patients, clinicians and embryologists as scheduling of embryo transfer is outlined. Additional studies are needed to confirm these correlations and to better define the role of baseline serum AMH level in the prediction of blastocyst formation.

Recipient screening in IVF: first data from women undergoing anonymous oocyte donation in Dublin.

BACKGROUND: Guidelines for safe gamete donation have emphasised donor screening, although none exist specifically for testing oocyte recipients. Pre-treatment assessment of anonymous donor oocyte IVF treatment in Ireland must comply with the European Union Tissues and Cells Directive (Directive 2004/23/EC). To determine the effectiveness of this Directive when applied to anonymous oocyte recipients in IVF, we reviewed data derived from selected screening tests performed in this clinical setting. METHODS: Data from tests conducted at baseline for all women enrolling as recipients (n = 225) in the anonymous oocyte donor IVF programme at an urban IVF referral centre during a 24-month period were analysed. Patient age at programme entry and clinical pregnancy rate were also tabulated. All recipients had at least one prior negative test for HIV, Hepatitis B/C, chlamydia, gonorrhoea and syphilis performed by her GP or other primary care provider before reproductive endocrinology consultation. RESULTS: Mean (±SD) age for donor egg IVF recipients was 40.7 ± 4.2 yrs. No baseline positive chlamydia, gonorrhoea or syphilis screening results were identified among recipients for anonymous oocyte donation IVF during the assessment interval. Mean pregnancy rate (per embryo transfer) in this group was 50.5%. CONCLUSION: When tests for HIV, Hepatitis B/C, chlamydia, gonorrhoea and syphilis already have been confirmed to be negative before starting the anonymous donor oocyte IVF sequence, additional (repeat) testing on the recipient contributes no new clinical information that would influence treatment in this setting. Patient safety does not appear to be enhanced by application of Directive 2004/23/EC to recipients of anonymous donor oocyte IVF treatment. Given the absence of evidence to quantify risk, this practice is difficult to justify when applied to this low-risk population.