Decidualization score identifies an endometrial dysregulation in samples from women with recurrent pregnancy losses and unexplained infertility.

OBJECTIVE: To study decidualization-associated endometrial factors. DESIGN: Retrospective cohort study to compare endometrial gene expression patterns in women experiencing reproductive failure including recurrent pregnancy loss or unexplained infertility versus fertile controls. SETTING: University Reproductive Medicine Center. PATIENTS: Women experiencing recurrent reproductive failure including recurrent pregnancy loss or unexplained infertility (n = 42) and fertile controls (n = 18). INTERVENTIONS: Endometrial biopsy samples were analyzed with targeted ribonucleic acid sequencing via next-generation sequencing. MAIN OUTCOME MEASURES: The primary end point measurements were the expression of genes important for endometrial transformation during decidualization measured singly and in a combined/cumulative score approach. The secondary end point measurements were receiver operating curve analysis and comparisons between the specific biomarkers. RESULTS: The comparison revealed differential expression of factors associated with decidualization, tissue homeostasis, and immune regulation: FOXO1, GZMB, IL15, SCNN1A, SGK1, and SLC2A1. A combined evaluation of these 6 signature factors was designated as a decidualization score in which the maximal score was "6" and the minimal was "0". Among controls, 89% of the samples had a score ≥5 and 11% had a score of "4". A total of 76% of samples in the patient group had scores ≤4 and 19% had the lowest score of "0". A decidualization score <4 provided evidence of abnormality in the decidualization process with a sensitivity of 76% (95% CI 61%-88%) and specificity of 89% (95% CI 65%-99%). CONCLUSIONS: Decidualization scoring can determine whether the endometrial molecular profile is implantation-friendly. Further validation of this testing approach is necessary to determine a particular patient population in whom it could be used for selecting patients that require therapeutic actions to improve endometrial conditions prior to the in vitro fertilization procedure.

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.

COVID-19 and immunomodulation treatment for women with reproductive failures.

COVID-19 pandemic is affecting various areas of health care, including human reproduction. Many women with reproductive failures, during the peri-implantation period and pregnancy, are on the immunotherapy using immune modulators and immunosuppressant due to underlying autoimmune diseases, cellular immune dysfunction, and rheumatic conditions. Many questions have been raised for women with immunotherapy during the COVID-19 pandemic, including infection susceptibility, how to manage women with an increased risk of and active COVID-19 infection. SARS-CoV-2 is a novel virus, and not enough information exists. Yet, we aim to review the data from previous coronavirus outbreaks and current COVID-19 and provide interim guidelines for immunotherapy in women with reproductive failures.

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.

Is superfertility associated with recurrent pregnancy loss?

PROBLEM: A recent hypothesis has implicated superfertility as a cause of recurrent pregnancy loss. Clinical support for the concept comes from one report that 40% of women experiencing recurrent miscarriages had monthly fecundity rates of 60% or greater and thus were designated as superfertile. METHODS OF STUDY: To confirm or refute this finding, clinical histories of 201 women with a history of recurrent pregnancy loss were reviewed and months to desired pregnancy, karyotypes of their products of conception as well as results of laboratory tests including antiphospholipid antibodies and circulating natural killer cells were recorded. RESULTS: The prevalence of superfertility was 32% (64/201) among recurrently aborting women compared with 3% of the general population according to the model of Tietze (P < 0.0001). Fifty-nine of the 201 (30%) study patients displayed presence of APA,LA, increased CD56(+) cells, or increased NK cytotoxicity and were designated as having an immunologic risk factor. Of the 192 karyotypes of products of conception from women with a history of recurrent miscarriage, 153 (80%) had a normal chromosome complement and 38 (20%) were abnormal. Among the normal karyotypes, 86 (56%) were 46XX and 67 (44%) were 46XY. CONCLUSION: Recurrent pregnancy loss is associated with superfertility in 32%, immunologic risk factors in 30% and a 20% frequency of chromosomally abnormal pregnancy losses. Thus, implantation failure can result from too much or too little implantation.

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.

Is apolipoprotien E codon 112 polymorphisms associated with recurrent pregnancy loss?

PROBLEM: To compare the prevalence of 112T>C point mutations among women experiencing RPL with fertile control women. METHOD OF STUDY: Buccal swabs were obtained from 232 individuals: 136 with a history of >or=2 abortions, 37 with at least 2 live births and 59 with a history of deep vein thrombosis (DVT). DNA was extracted and PCR amplification of Apo E codons was performed. RESULTS: The allelic frequency of a cytosine at position 112 was 11.4% (31/272) among patients experiencing RPL, compared with a frequency of 5.4% (4/74) among the fertile controls (P = 0.19) and 19.5% (23/118) among individuals with a history of DVT. However, significantly more E3/E4 and E4/E4 genotypes were seen among individuals experiencing RPL and DVT than fertile controls (P < 0.05). CONCLUSION: Apo E4 codon 112C point mutation is, by itself, not associated with an elevated risk of recurrent pregnancy loss, but rather codon 112C in association with codon 158C is a risk factor for RPL.

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.

Are polymorphisms in the ACE and PAI-1 genes associated with recurrent spontaneous miscarriages?

PROBLEM: To determine whether the ACE D/D genotype or the combination of PAI-1 4G/4G and ACE D/D genotypes may serve as a risk factor for recurrent pregnancy loss. METHOD OF STUDY: Buccal swabs were obtained from 120 women experiencing recurrent pregnancy loss and from 84 fertile control women. DNA was extracted from the buccal swab samples using the Qiagen DNA Mini Kit (Qiagen), followed by multiplex polymerase chain reaction (PCR). PCR products were analyzed for the ACE gene polymorphism, which consists of the insertion or deletion (I/D) of a 287-bp fragment in intron 16, and the PAI-1 4G/4G genotype. RESULTS: No significant differences in specific ACE gene mutations were observed when patients experiencing recurrent miscarriage were compared with control women. When the frequencies of homozygous mutations for ACE D/D and PAI-I 4G/4G were compared between recurrent aborters and controls, again no significant differences in the prevalence of the combination of these gene mutations were noted. CONCLUSION: Homozygosity for the D allele of the ACE gene and the combination of the D/D genotype with two 4G alleles of the PAI-1 promoter gene are not associated with a significant increase in the risk of recurrent miscarriage.

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.

The association of Apoprotein E polymorphisms with recurrent pregnancy loss.

PROBLEM: We have previously reported the role of polymorphisms of thrombogenic genes involved in coagulation and fibrinolysis as risk factors for recurrent pregnancy loss. Thrombophilia has been viewed as a multigenic disorder rather than a monogenetic clinical phenotype and Apo E has been shown to play an important role in lipid metabolism in pregnancy. As individuals carrying the E4 allele of the ApoE gene have the highest risk for thrombosis, we evaluated the frequency of the Apo E4 genotype among women suffering from recurrent pregnancy loss. METHOD OF STUDY: Buccal swabs were obtained from 69 women with a history of two or more consecutive spontaneous abortions and 37 women with at least two live births and not more than one miscarriage. DNA was extracted from the buccal swabs and PCR amplification of Apo E2, E3, and E4 was performed. RESULTS: Women experiencing recurrent pregnancy loss had a significantly higher prevalence of Apo E3/4, E4/4 genotypes (21.7%) compared with control women (5.4%) (P = 0.036). CONCLUSION: Apo E4 polymorphism may contribute to the thrombophilic risk factors contributing to recurrent pregnancy loss.