Intralipid treatment for women with reproductive failures.

Does intravenous intralipid treatment for reproductive failure enhance live births? The answer to this question is yes, BUT only in patients that have a diagnosis of recurrent implantation failure (RIF) or recurrent pregnancy loss (RPL) loss AND demonstrate elevated NK (natural killer) cell density in their endometrial biopsy. Live birth rates have been reported between 33% and 42% among women displaying elevated NK activity with a diagnosis of RIF and 75% and 91% among women experiencing RPL after intralipid infusion. When the pregnancy outcomes of women with a history of reproductive failure and elevated NK cells treated with intralipid were evaluated, the overall live birth rate per cycle of treatment was 61%. The results of published studies suggest that intralipid can be used successfully as a therapeutic option to modulate abnormal NK activity in women with reproductive problems.

Prevalence of HHV-6 in endometrium from women with recurrent implantation failure.

PROBLEM: To study the prevalence of HHV-6 in endometrial biopsies among women experiencing recurrent implantation failure (RIF) after IVF/ET compared with controls. METHOD OF STUDY: Thirty women experiencing RIF after IVF/ET and 10 fertile women participated in the study. All women had endometrial biopsies taken in the luteal phase of their menstrual cycle for an endometrial immune profile (EIP) and HHV-6 mRNA as well as lymphocyte and granulocyte populations. The prevalence of HHV-6 in endometrial biopsies was determined, and biopsies for positive and negative expression of HHV-6 were compared with the results of their EIP and lymphocyte and granulocyte populations. RESULTS: Thirty-seven percentage of women with a history of RIF and 0% of controls demonstrated the presence of HHV-6 in their endometrial biopsies. No associations were found when the results of the endometrial immune profile were compared with the presence or absence of HHV-6. Significant increase in neutrophil-specific CD16b mRNA was found in HHV-6-positive samples, and the levels of B cells-related CD19 mRNA were lower in biopsies from women with RIF in comparison with normal controls. CONCLUSION: HHV-6 infection is an important factor in RIF.

Correction: PreImplantation factor (PIF) protects cultured embryos against oxidative stress: relevance for recurrent pregnancy loss (RPL) therapy.

[This corrects the article DOI: 10.18632/oncotarget.16028.].

PreImplantation factor (PIF) protects cultured embryos against oxidative stress: relevance for recurrent pregnancy loss (RPL) therapy.

Recurrent pregnancy loss (RPL) affects 2-3% of couples. Despite a detailed work-up, the etiology is frequently undefined, leading to non-targeted therapy. Viable embryos and placentae express PreImplantation Factor (PIF). Maternal circulating PIF regulates systemic immunity and reduces circulating natural killer cells cytotoxicity in RPL patients. PIF promotes singly cultured embryos' development while anti-PIF antibody abrogates it. RPL serum induced embryo toxicity is negated by PIF. We report that PIF rescues delayed embryo development caused by <3 kDa RPL serum fraction likely by reducing reactive oxygen species (ROS). We reveal that protein disulfide isomerase/thioredoxin (PDI/TRX) is a prime PIF target in the embryo, rendering it an important ROS scavenger. The 16F16-PDI/TRX inhibitor drastically reduced blastocyst development while exogenous PIF increased >2 fold the number of embryos reaching the blastocyst stage. Mechanistically, PDI-inhibitor preferentially binds covalently to oxidized PDI over its reduced form where PIF avidly binds. PIF by targeting PDI/TRX at a distinct site limits the inhibitor's pro-oxidative effects. The >3kDa RPL serum increased embryo demise by three-fold, an effect negated by PIF. However, embryo toxicity was not associated with the presence of putative anti-PIF antibodies. Collectively, PIF protects cultured embryos both against ROS, and higher molecular weight toxins. Using PIF for optimizing in vitro fertilization embryos development and reducing RPL is warranted.

A search to identify genetic risk factors for endometriosis.

PROBLEM: To search for molecular markers of endometriosis the following polymorphisms: p53 codon 72 Pro (apoptosis), TNF alpha-308 (inflammation), VEGF-1164AA (angiogenesis), and SOD2 (oxidative stress) were investigated. METHOD OF STUDY: Forty-two women-24 with surgically proven endometriosis and 18 with no endometriosis found at the time of laparoscopy-had buccal swabs taken for DNA analyses of 4 gene polymorphisms including p53codon72, TNF-308 G/A, VEGF-1154G/A, SOD Ala16Val DNA. The frequencies of genotypes and alleles of these polymorphisms were compared between women with and without endometriosis. RESULTS: No specific gene mutation differences for the four genes tested nor differences in the frequencies of heterozygous and homozygous mutations were found between patients with endometriosis and controls. In addition, no differences in allelic frequencies of the four genetic polymorphisms were observed between patients with endometriosis and control. CONCLUSION: Endometriosis is not associated with gene mutations for p53codon72, TNF-308 G/A, VEGF-1154G/A, SOD Ala16Val.

Preimplantation factor inhibits circulating natural killer cell cytotoxicity and reduces CD69 expression: implications for recurrent pregnancy loss therapy.

Embryo-secreted preimplantation factor (PIF) is necessary for, and its concentration correlates with, embryo development in humans by promoting implantation and trophoblast invasion. Synthetic PIF (sPIF) modulates systemic immunity and is effective in autoimmune disease models. sPIF binds monocytes and activated T and B cells, leading to immune tolerance without suppression. This study examined the effect of sPIF on natural killer (NK) cell cytotoxicity in 107 consecutive nonselected, nonpregnant patients with recurrent pregnancy loss (RPL) and 26 infertile IVF patients (controls). The effects of sPIF, intravenous gamma immunoglobulin (Ig), Intralipid and scrambled PIF (PIFscr; negative control) on NK cell cytotoxicity to peripheral-blood cells were compared by flow cytometry of labelled-K562 cell cytolysis. The effects of sPIF and PIFscr on whole-blood NKCD69+ expression were also compared. In patients with RPL, sPIF inhibited NK cell cytotoxicity at doses of 2.5 and 25ng/ml (37% and 42%) compared with PIFscr (18%; P<0.001), regardless of the proportion of peripheral-blood NKCD56+ cells to lymphocytes. Pre-incubation of blood from infertile patients with sPIF for 24h decreased NKCD69+ expression versus incubatino with PIFscr (P<0.05). In conclusion, sPIF inhibits NK cell cytotoxicity by reducing NKCD69 expression, suggesting a significant role in RPL patients. There is a continuous search to identify safe and effective agents to counteract recurrent pregnancy loss (RPL). Preimplantation factor (PIF) secreted by the embryo at the 2-cell stage is present throughout viable pregnancy but absent in nonviable pregnancy. Its immunomodulatory (not suppressive) effects promote embryo acceptance and maintenance by mother/host, control inflammation, facilitate uterine environment and placental embedding. Synthetic PIF (sPIF) was used to complete PIF's role as a targeted, safe treatment for immune-based RPL. Previous reports showed sPIF's significant protective systemic effect against maternal factors present in RPL serum. Herein is examined sPIF's ability to inhibit the local protective toxicity induced by natural killer (NK) immune cells in a representative number of RPL patients. When elevated in blood, NK cells are associated with RPL. Low-dose physiological sPIF was highly effective to inhibit NK cell toxicity. Side-by-side comparison showed that sPIF is equally effective at a lower dose than intravenous gamma immunoglobulin or Intralipid treatment currently used. The sPIF effect on NK cells was targeted, indicating specific action. Overall, sPIF may represent a safe, effective and nontoxic immune-based therapy against RPL.

Does immunotherapy for treatment of reproductive failure enhance live births?

Before effective treatment for reproductive failure can be instituted, the cause of the failure must be determined. A search of PubMed was made to identify the published data regarding diagnosis and treatment of reproductive failure. Results were compared with the frequency of antiphospholipid antibodies (APA) in 2995 women with histories of unexplained infertility, recurrent implantation failure, recurrent pregnancy loss, and fertile women. In addition, pregnancy outcomes among 442 women experiencing reproductive failure and elevated NK cell activity after treatment with intravenous immunoglobulin (IVIg) (N = 242) or intralipids (N = 200) were compared. The prevalence of APA was the same among women with the diagnosis of unexplained infertility, recurrent implantation failure, and recurrent miscarriage. Heparin and aspirin are successful in the treatment of elevated APA among women with recurrent miscarriage but not with recurrent implantation failure. IVIg has been successful in the treatment of recurrent miscarriage and recurrent implantation failure among women with elevated APA and/or NK cell activity. When the pregnancy outcomes of women with a history of reproductive failure and elevated NK cell cytotoxicity treated with intralipid were compared with women treated with IVIg, no differences were seen. Immunotherapy for treatment of reproductive failure enhances live birth but only in those women displaying abnormal immunologic risk factors.

Preimplantation factor negates embryo toxicity and promotes embryo development in culture.

Preimplantation factor (PIF) is secreted by viable mammalian embryos and promotes implantation and trophoblast invasion. Whether PIF also has a direct protective or promoting effect on the developing embryo in culture is unknown. This study examined the protective effects of synthetic PIF (sPIF) on embryos cultured with embryo toxic serum (ETS) from recurrent pregnancy loss patients (n=14), by morphological criteria at 72 h of culture, and determined sPIF-promoting effect on singly cultured bovine IVF embryo development. sPIF negated the ETS-induced effect by increasing mouse blastocyst rate versus other embryonic stages (odds ratio (OR) 2.01, 95% confidence intervals (CI) 1.14-3.55, chi-squared=12.74, P=0.002), increased blastocyst rate (39.0% versus 23.7% ETS alone) and lowered embryo demise rate (11.0% versus 28.8%, OR 0.24, 95% CI 0.11-0.54), which was not replicated by scrambled PIF or the control. sPIF added to bovine embryos for 3 days promoted development at day 7 of culture (11% versus 0%, chi-squared=4.0, P=0.045). In conclusion, sPIF prevented embryo demise caused by exposure to ETS and promoted development of singly cultured bovine IVF embryos following short-term exposure. sPIF-based therapy for reducing recurrent pregnancy loss and improving lagging cultured IVF embryo development should be explored.

PreImplantation Factor (PIF) correlates with early mammalian embryo development-bovine and murine models.

BACKGROUND: PreImplantation Factor (PIF), a novel peptide secreted by viable embryos is essential for pregnancy: PIF modulates local immunity, promotes decidual pro-adhesion molecules and enhances trophoblast invasion. To determine the role of PIF in post-fertilization embryo development, we measured the peptide's concentration in the culture medium and tested endogenous PIF's potential trophic effects and direct interaction with the embryo. METHODS: Determine PIF levels in culture medium of multiple mouse and single bovine embryos cultured up to the blastocyst stage using PIF-ELISA. Examine the inhibitory effects of anti-PIF-monoclonal antibody (mAb) added to medium on cultured mouse embryos development. Test FITC-PIF uptake by cultured bovine blastocysts using fluorescent microscopy. RESULTS: PIF levels in mouse embryo culture medium significantly increased from the morula to the blastocyst stage (ANOVA, P = 0.01). In contrast, atretic embryos medium was similar to the medium only control. Detectable - though low - PIF levels were secreted already by 2-cell stage mouse embryos. In single bovine IVF-derived embryos, PIF levels in medium at day 3 of culture were higher than non-cleaving embryos (control) (P = 0.01) and at day 7 were higher than day 3 (P = 0.03). In non-cleaving embryos culture medium was similar to medium alone (control). Anti-PIF-mAb added to mouse embryo cultures lowered blastocyst formation rate 3-fold in a dose-dependent manner (2-way contingency table, multiple groups, X2; P = 0.01) as compared with non-specific mouse mAb, and medium alone, control. FITC-PIF was taken-up by cultured bovine blastocysts, but not by scrambled FITC-PIF (control). CONCLUSIONS: PIF is an early embryo viability marker that has a direct supportive role on embryo development in culture. PIF-ELISA use to assess IVF embryo quality prior to transfer is warranted. Overall, our data supports PIF's endogenous self sustaining role in embryo development and the utility of PIF- ELISA to detect viable embryos in a non-invasive manner.

Prevalence of antiphospholipid antibodies among women experiencing unexplained infertility and recurrent implantation failure.

The prevalences of antiphospholipid antibodies (APAs) among 1,325 women with a history of unexplained infertility and 676 women experiencing recurrent implantation failure were compared with 789 women experiencing recurrent pregnancy loss and 205 fertile control women. Eight percent and 9% of women with a history of unexplained infertility and recurrent implantation failure had more than one positive APA compared with 1.5% of fertile negative control women and 11% of positive control women experiencing recurrent pregnancy loss.

How to predict implantation? No correlation between embryonic aneuploidy and soluble human leukocyte antigen G-concentrations.

OBJECTIVE: To determine if soluble human leukocyte antigen-G (sHLA-G) concentrations in spent culture media may assist in identifying the normal embryo for implantation. DESIGN: Prospective blinded comparative study. SETTING: Reproductive genetic and reproductive medicine centers. PATIENT(S): One hundred and sixteen embryos obtained from eight patients undergoing in vitro fertilization (IVF) with preimplantation genetic diagnosis (PGD). INTERVENTION(S): Culture media obtained 2 days after fertilization were analyzed for sHLA-G concentrations using an enzyme-linked immunosorbent assay (ELISA) assay. A sHLA-G concentration of >or=1.9 mIU/mL was considered a positive predictor for successful implantation. Polar bodies and blastomeres from day-3 embryos were tested by PGD for 5 to 11 chromosomes: 8, 9, 13, 15, 16, 17, 18, 21, 22, X, and Y. MAIN OUTCOME MEASURE(S): The results of the sHLA-G concentrations were compared with the results of the PGD analyses. RESULT(S): We found an sHLA-G concentration >or=1.9 mIU/mL in 48% (56 out of 116) and normal PGD results in 52% (57 out of 116) of embryos. Of the embryos with normal PGD results, 46% (26 out of 57) had sHLA-G concentrations >or=1.9 mIU/mL. Among the embryos with sHLA-G >or=1.9 mIU/mL, 46% (26 out of 56) had normal PGD results, and 21% of embryos displayed both normal PGD results and sHLA-G >or=1.9 mIU/mL. CONCLUSION(S): No correlation between concentrations of sHLA-G in embryo culture media and PGD results of an embryo's aneuploidy were observed.

Thrombophilic gene polymorphisms are risk factors for unexplained infertility.

Earlier studies have shown inherited thrombophilia to be a risk factor for recurrent implantation failure, raising the question of whether this risk is related to the underlying cause of unexplained infertility or to the mechanisms involved in the implantation process. When nine thrombophilic gene polymorphisms were compared among 92 women with the diagnosis of unexplained infertility and 60 fertile control women, women with a history of unexplained infertility displayed a higher prevalence of MTHFR C677T polymorphisms than control women.

Interleukin 1 receptor antagonist gene polymorphisms are not risk factors for recurrent pregnancy loss: evaluation of couples.

PROBLEM: The interleukin-1 system has been implicated in pregnancy outcome. Fetal carriage of interleukin-1 receptor antagonist (IL-1Ra) specific alleles has been associated with adverse pregnancy outcomes including spontaneous abortion and pre-term labor. This study was undertaken to compare the frequency of IL-1RN 2 alleles among both male and female partners of couples experiencing recurrent pregnancy loss with that of fertile control couples. METHOD OF STUDY: Buccal swabs were obtained from 42 couples experiencing recurrent pregnancy loss and from 20 fertile control couples. DNA was extracted from the buccal swabs and analyzed for the presence of IL-1RN variable number tandem repeat. RESULTS: No significant differences were found when the frequency of IL-1RN 2 polymorphisms were compared between fertile control couples and couples experiencing recurrent pregnancy loss. Similar results were also obtained when comparing women or men respectively from each group. CONCLUSION: IL-1RN 2 allele is not a risk factor for recurrent pregnancy loss.

Duration of intralipid's suppressive effect on NK cell's functional activity.

BACKGROUND: In vitro investigations have revealed the ability of intralipids to suppress natural killer (NK) cytotoxicity. Evidence from both animal and human studies suggests that intralipid administered intravenously may enhance implantation and maintenance of pregnancy when the patient has an abnormal NK cell level or function. PROBLEM: The aim of this study was to establish the duration and efficacy of Intralipids suppressive effect on NK cell functional activity. METHOD OF STUDY: Fifty patients with abnormal NK activity results (NKa) received intralipid 20% i.v. (9 mg/mL total blood volume -corresponds to 2 mL of intralipid 20% diluted in 250 mL saline; or 18 mg/mL - corresponds to 4 mL of intralipid 20% diluted in 250 mL saline) infusions and their NKa were tested periodically. The determination of NK cell function was performed by flow cytometry using K562 cells as targets. RESULTS: Fifty women with abnormal NKa-testing received intralipid infusions. 39 (78%) showed NKa suppression within the normal range the first week after infusion, 11 (22%), showed suppression, but still above the normal threshold. They received second infusion 2-3 weeks later. In 10, the Nka activity was normalized the following week. Four patients had three intralipid infusions in 2-week periods in between and after the third infusion, and all showed NKa normal activity. In 47 patients the suppressive effect of the Intralipid after the normalization of NKa lasted between 6 and 9 weeks, in two patients this benefit lasted 5 weeks, and in one patient the effect was 4 weeks. CONCLUSION: Intralipid is effective in suppressing in vivo abnormal NK-cell functional activity. The results suggest that Intralipid can be used successfully as a therapeutic option to modulate abnormal NK activity in women with reproductive failure.

Comparison of thrombophilic gene mutations among patients experiencing recurrent miscarriage and deep vein thrombosis.

PROBLEM: Inherited thrombophilia has been shown to be a risk factor for cardiovascular disease including deep venous thrombosis as well as reproductive disorders including recurrent pregnancy loss. We have previously reported three out of the 10 thrombophilic mutations studied, plasminogen activator inhibitor-1 (PAI-1) 4G/5G, factor XIII V34L, and homozygous MTHFR C667T, correlated significantly with recurrent pregnancy loss compared with controls. This study was undertaken to compare the frequencies of nine inherited thrombophilias among women with a history of recurrent pregnancy loss with individuals experiencing deep venous thrombosis and fertile controls. METHOD OF STUDY: Six hundred thirty-four participants including 550 women with a history of recurrent pregnancy loss, 43 individuals with deep vein thrombosis and 41 fertile women without a history of recurrent miscarriage. All participants had buccal swabs taken for DNA analyses of nine gene polymorphisms including factor V G1691A, factor V H1299R (R2), factor II Prothrombin G20210A, factor XIII V34L, beta-fibrinogen -455G>A, PAI-1 4G/5G, human platelet antigen 1 a/b (L33P), MTHFR C677T, MTHFR A1298C. Frequencies of thrombophilic gene polymorphisms were compared among the three populations studied. RESULTS: Individuals with a history of DVT had a significantly higher frequency of all of the polymorphisms studied compared with women experiencing a history of recurrent pregnancy loss and the fertile controls. The frequencies of mutations for V34L and PAI-1 4G/5G were significantly increased among women experiencing recurrent pregnancy loss compared with controls. The most prevalent polymorphisms were factor XIII V34L and PAI-1 4G/4G for both individuals with a history of deep vein thrombosis and recurrent pregnancy loss compared with controls. CONCLUSION: Screening for risk factors for inherited thrombophilia with only polymorphisms for factor V von Leiden, factor II prothrombin and MTHFR may be missing the more prevalent identifiers of jeopardy.

Association of progesterone receptor polymorphisms with recurrent implantation failure after in vitro fertilization and embryo transfer.

INTRODUCTION: Progesterone is the hormone of pregnancy and is required for its initiation. The actions of progesterone are mediated by the progesterone receptor. Polymorphic variants of human progesterone receptor genes have been implicated in implantation failure. MATERIALS AND METHODS: We, therefore, investigated the prevalence of H770H (C/T genotype), V660L polymorphism and a 306 bp Alu insertion in exon 7 of the progesterone receptor among women with history of recurrent implantation failure to determine whether any of these polymorphisms may serve as a risk factor for implantation failure. DNA was extracted from the buccal swabs obtained from 66 women experiencing implantation failure and 75 fertile control women. PCR amplification of fragments was purified and the DNA sequenced to identify the polymorphism. The frequencies for the three variants were 27% for H770H, 21% for V660L and 0% for the 306 bp Alu insertion in exon 7 among women with implantation failure compared with control women of 25% for H770H and 24%for V660L and 0% for the 306 bp Alu insertion in exon 7. DISCUSSION: No significant differences in the overall allelic frequency of progesterone receptor variants was seen when women experiencing recurrent implantation failure were compared with control women. CONCLUSION: We conclude that the H770H and V660L and PROGINS progesterone receptor polymorphisms are not markers that can identify women at risk for recurrent implantation after IVF/ET.

Cryopreservation of human sperm in a lecithin-supplemented freezing medium.

The use of egg yolk and serum albumin as additive diluents for human sperm cryopreservation is routine. But because both diluents are of animal origin, they potentially may introduce microbial agents to the sample. To reduce the risk of contamination, the cryoprotective property of phospholipids extracted from lecithin was evaluated and found to be effective when supplemented with dimethyl sulfoxide and glycerol.

Vascular endothelial growth factor gene polymorphisms and recurrent pregnancy loss.

PROBLEM: To be successful, pregnancy must induce its own blood supply through angiogenesis and vascular endothelial growth factor (VEGF) is the best characterized regulator of angiogenesis and one polymorphism of the VEGF gene, -1154, has been suggested to be associated with recurrent spontaneous abortion. The aim of this study was to confirm or refute the relationship of VEGF -1154 to recurrent pregnancy loss (RPL). METHOD OF STUDY: Buccal swabs were obtained from 152 women with history of two or more consecutive spontaneous abortions and 65 control women. DNA was extracted from the buccal swabs and analyzed for the presence of the VEGF -1154A/A gene. RESULTS: The frequency of homozygosity of the VEGF -1154A gene was significantly higher among women experiencing RPL compared with fertile control women (16% versus 6%, P < 0.05). CONCLUSION: Homozygosity of the VEGF -1154A gene may serve as a susceptibility factor affecting for RPL.

HLA-G and its role in implantation (review).

BACKGROUND: Human leukocyte antigen G (HLA-G) is thought to play a key role in implantation by modulating cytokine secretion to control trophopblastic cell invasion and to maintain a local immunotolerance. METHOD OF STUDY: The literature is reviewed to provide a description of the genetic background, properties of the protein, and the function of HLA-G. Data are presented on potential clinical applications of HLA-G including the use of evaluation of HLA-G gene polymorphisms in the diagnosis of patients experiencing recurrent pregnancy loss and evaluation and testing of soluble HLA-G (sHLA-G) in embryo culture media for the selection of embryos for transfer after in vitro fertilization (IVF). RESULTS: The literature supports a central role of HLA-G for successful implantation. Of couples experiencing recurrent pregnancy loss, 32% demonstrated the -1725G HLA-G polymorphism. Our data showed that when embryos were selected for transfer after IVF based on culture media concentrations of sHLA-G > or = 2 U/ml and good morphologic grade, a 65% pregnancy rate compared with a 0% pregnancy rate in those with <2 U/ml sHLA-G. CONCLUSIONS: HLA-G is important for successful implantation in human beings. The HLA-G -725 promoter polymorphism is a risk factor for recurrent miscarriage. Measurement of sHLA-G in embryo culture media can help select embryos for transfer after IVF allowing fewer embryos to be transferred in an attempt to lower multiple gestation rates.