Intrauterine Administration of Activated Peripheral Blood Mononuclear Cells in Intrauterine Insemination: A Prospective Double-Blind Randomized Clinical Trial.

OBJECTIVE: To evaluate the effect of intrauterine administration of activated peripheral blood mononuclear cells (PBMC) on intrauterine insemination (IUI) success rates. METHODS: This prospective double-blind randomized parallel clinical trial included 213 patients undergoing IUI at the Fertilys clinic. PBMC were isolated on the day of ovulation (day 0; D0) and stimulated with phytohemagglutinin (PHA) and human chorionic gonadotropin (hCG) for 48 hours (day 2; D2). Patients in the PBMC group (n = 108) underwent in utero administration of 1.106 cells on D2, while patients in the control group (n = 105) were administered sperm-washing medium. Distribution of CD4 T lymphocyte populations (n = 61) was assessed on D0 and D2. Pregnancy and live birth rates were also evaluated. RESULTS: Demographic and clinical characteristics, pregnancy rates, and live birth rates were not significantly different between the PBMC and control groups. Significantly higher levels of T helper (Th) 2, Th22, and T regulatory cells (P < 0.0001) and lower levels of Th17 cells were observed in hCG-activated PBMC at D2 than at D0. CONCLUSION: Intrauterine administration of PBMC was not beneficial in IUI patients. New clinical approaches to better identify patients requiring endometrium immunomodulation needs to be addressed.

Should intrauterine inseminations still be proposed in cases of unexplained infertility? Retrospective study and literature review.

OBJECTIVE: Unexplained infertility is defined by the absence of identifiable causes of infertility. The results of randomized studies and meta-analysis regarding the treatment of unexplained infertility are discordant due to methodological problems. DESIGN: The aim of this study is to compare the clinical pregnancy rate per cycle (CPR/c) in IUI and IVF/ICSI in cases of unexplained infertility, according to the woman's age group and to identify the factors which predict success. INTERVENTIONS: We performed a retrospective study in two ART centers, comparing overall clinical pregnancy, ongoing pregnancy and live birth rates in IVF/ICSI and IUI. We also compared pregnancy and birth rates according to different female age groups. RESULTS: 855 IVF/ICSI and 804 IUI cycles were compared. We found a significant difference (p < 0.001) in the pregnancy and live birth rates per cycle between IUI and IVF/ICSI, overall and in the different female age groups, except in women aged 40 and over. The greatest chances of pregnancy with IUI are found in women with secondary unexplained infertility, during the first two cycles and with a bi-follicular response to stimulation. In IVF/ICSI, pregnancy rates are higher in women with secondary unexplained infertility, in the first two cycles, in IVF and in women receiving a transfer of two embryos regardless of the embryonic stage. CONCLUSION: We recommend IVF/ICSI treatment rather than IUI for unexplained infertility (OR CPR/c 4.20 with 95% CI [3.72-4.68]). This is in accordance with NICE, which advises the use of IVF after 2 years.

Impact of Endocrine Disruptors upon Non-Genetic Inheritance.

Similar to environmental factors, EDCs (endocrine-disrupting chemicals) can influence gene expression without modifying the DNA sequence. It is commonly accepted that the transgenerational inheritance of parentally acquired traits is conveyed by epigenetic alterations also known as "epimutations". DNA methylation, acetylation, histone modification, RNA-mediated effects and extracellular vesicle effects are the mechanisms that have been described so far to be responsible for these epimutations. They may lead to the transgenerational inheritance of diverse phenotypes in the progeny when they occur in the germ cells of an affected individual. While EDC-induced health effects have dramatically increased over the past decade, limited effects on sperm epigenetics have been described. However, there has been a gain of interest in this issue in recent years. The gametes (sperm and oocyte) represent targets for EDCs and thus a route for environmentally induced changes over several generations. This review aims at providing an overview of the epigenetic mechanisms that might be implicated in this transgenerational inheritance.

Endometrium Immunomodulation to Prevent Recurrent Implantation Failure in Assisted Reproductive Technology.

After more than four decades of assisted reproductive technology (ART) practice worldwide, today more than 60% of women undergoing in vitro fertilization (IVF) treatments fail to become pregnant after the first embryo transfer and nearly 20% of patients are suffering from unexplained recurrent implantation failures (RIFs) and repeated pregnancy loss (RPL). The literature reported different causes of RIF-RPL, mainly multifactorial, endometrial and idiopathic. RIF remains a black box because of the complicated categorization and causes of this physio-pathological dysregulation of implantation and pregnancy process after ovarian stimulation. Many options were suggested as solutions to treat RIF-RPL with controversial results on their usefulness. In this article, we reviewed different possible therapeutic options to improve implantation rates and clinical outcomes. Based on our experience we believe that endometrium immunomodulation after intrauterine insemination of activated autologous peripheral blood mononuclear cells (PBMCs) or platelet-rich plasma (PRP) can be a promising therapeutic solution. On the other hand, peripheral lymphocyte balance typing, specific cytokines and interleukins profiling can be proposed as predictive biomarkers of implantation before embryo transfer.

Circulating MMP-7 and VEGF as potential predictive biomarkers for recurrent implantation failures.

Recurrent implantation failure (RIF) is considered to be one of the major limiting factors of assisted reproductive technology (ART) programme success. The current study focused on the investigation of matrix metalloproteinases (MMPs), tissue inhibitors of MMPs (TIMPs), cytokines and cell adhesion molecules in peripheral blood (PB) and follicular fluid (FF) obtained from 44 women aged between 25 and 39 years old and undergoing intracytoplasmic sperm injection (ICSI). These women were divided into two groups: 22 RIF women with embryo implantation failures after the transfer of at least four fresh or frozen-thawed good quality embryos in a minimum of three ICSI cycles, and 22 ICSI success women (controls) who achieved a clinical pregnancy at their first ICSI attempt. The PB and FF samples were obtained from each patient on the day of oocyte retrieval. MMP-1, -2, -3, -7, -9, TIMP-1, -2, vascular endothelial growth factor (VEGF), leukaemia inhibitory factor (LIF), vascular cell adhesion molecule 1 (VCAM1) and intercellular adhesion molecules 1 (ICAM1) were analyzed using enzyme-linked immunosorbent assay of PB and FF. Our results showed significant decreases in PB MMP-7 and PB VEGF in the RIF group compared with controls [281.11 (33-614) pg/ml vs 119.92 (27-441) pg/ml; P-value = 0.030] and [82.54 (25.94-210.20) pg/ml vs 30.93 (13.62-193.33) pg/ml; P-value = 0.022; respectively]. Receiver operating characteristic (ROC) curve analysis showed informative area under the curve values for PB MMP-7, as well as for PB VEGF, making them able to be proposed as biomarkers of the RIF. Therefore, circulating MMP-7 and VEGF seem to play an interesting role in embryo implantation in in vitro fertilization (IVF)/ICSI cycles and could be proposed as circulating biomarkers of the RIF. These results could be helpful for clinicians and patients to choose the best rescue strategy and treatment to minimize implantation failure in women undergoing IVF/ICSI procedures after the first attempt.

Seminal cell-free DNA and sperm characteristic's: An added biomarker for male infertility investigation.

Cell-free DNA (Cf-DNA) fragments may constitute an easy-to-measure molecular tool for guiding the choice of care provided to infertile couples who benefit assisted reproductive technology (ART) programmes. Data on Cf-DNA levels in the seminal plasma of men with sperm alterations are scarce. The objective of the present study was to quantify the presence of Cf-DNA in semen by using a quantitative real-time PCR. We compared men with abnormal sperm characteristics (n = 21) with normospermic controls (n = 21). The PCR assay evidenced significantly higher mean Cf-DNA levels in patients with sperm abnormalities than in controls (2.09 versus 1.18 µg/ml, respectively; p = .0003). The Cf-DNA levels were notably higher in men with azoospermia (3.65 µg/ml, versus 1.34 µg/ml in matched controls; p = .03) and men with teratozoospermia (1.80 µg/ml, versus 1.29 µg/ml in matched controls; p = .008). Our data report a significant association between elevated Cf-DNA levels and sperm abnormalities. These results may open up new diagnostic and prognostic perspectives in male infertility.

Decline in semen quality of North African men: a retrospective study of 20,958 sperm analyses of men from different North African countries tested in Tunisia over a period of 6 years (2013-2018).

BACKGROUND: According to numerous studies from around the world, semen quality seems to have declined dramatically in recent times. However, the data available on male fertility status and semen quality in North Africa are limited. AIM: To investigate the status of semen quality in North-African men and to understand its variations. SUBJECTS & METHODS: 20,958 sperm-analyses (Spermogram - Spermocytogram) of North-African men (19-77 years old) consulting for infertility, performed in a private laboratory of medical analyses (Tunis, Tunisia) over a period of 6 years (2013-2018), were investigated. All patients had at least 1 year of unprotected intercourse with their partners before the test. Statistical analyses were performed using SPSS 22.0 software for windows. RESULTS: Libyan men presented a clear decline in all sperm parameters. A continuous decline in sperm morphology quality was shown in Tunisian and Algerian men. Mauritanian men presented a significant increase in sperm vitality with pseudo-stability in the rest of the sperm parameters during the whole study period. CONCLUSION: North-African men presented remarkable decreases in their semen quality over the last decade. This data could confirm possible global common-causes that need to be identified in order to limit their negative impact on sperm quality, and consequently on male-fertility.

CENTRAL RETINAL VEIN OCCLUSION IN OTHERWISE HEALTHY CHILDREN AND ADOLESCENTS: Association With Multigenetic Variants of Thrombophilia.

PURPOSE: To report cases of central retinal vein occlusion in otherwise healthy children showing combined genetic variants of thrombophilia. METHODS: Ophthalmological, pediatric records and genetic analyses of thrombophilia-associated variants were retrospectively reviewed in four children diagnosed with central retinal vein occlusion. Genetic screening, including Factor XII, platelet glycoprotein (GP) IIIa PlA1/A2 (rs5918), and GPIa/IIa C807T (rs1126643) and G873A (rs1062535) mutations, was performed by PCR amplification and Sanger sequencing of PCR products. The genotyping of prothrombin G20210A, Leiden Factor V G1691A, methylenetetrahydrofolate reductase (MTHFR) C677T/A1298C mutations, and plasminogen activator inhibitor-1 4G/5G polymorphisms was performed by real-time PCR with Fluorescence Resonance Energy Transfer (FRET) probes. RESULTS: The genotyping analysis identified combined genetic variants of thrombophilia in each patient. Mutations for MTHFR (C677T) and GPIIIa PlA1/A2 were detected in Case 1, mutations for MTHFR (C677T), GPIIIa PlA1/A2, and GPIa/IIa in Case 2, mutations for MTHFR (C677T) and GPIa/IIa in Case 3, and mutation for MTHFR (A12986C), GPIIIa Pl A1/A2, and GPIa/IIa in Case 4. Preventive low-dose aspirin therapy was prescribed to all patients. During a follow-up of 5 and 8 years, neither central retinal vein occlusion recurrence nor any other thrombotic event was observed in Cases 1 and 2, respectively. CONCLUSION: In otherwise healthy children presenting central retinal vein occlusion, genetic investigations for thrombophilia-associated variants should be considered, given the possible long-term benefit of aspirin prophylaxis.

Pregnancy after oocyte donation in a patient with NLRP7 gene mutations and recurrent molar hydatidiform pregnancies.

Molar pregnancies are benign trophoblastic diseases associated with a risk of malignant transformation. If aetiology remains mostly unknown, the risk of recurrent molar pregnancy is around 1.5% after one molar pregnancy and around 25% after 2 molar pregnancies. In the later situation, genetic mutations have been described, increasing hugely this risk. In case of mutations, probability to obtain a normal pregnancy is estimated around 1.8%. We report the case of a Caucasian 30-year-old woman whose previous five spontaneous pregnancies had a negative outcome: a spontaneous miscarriage and then 4 complete hydatidiform moles. Genetic testing revealed that the patient carried two heterozygous mutations in the NLRP7 gene (c.2982-2A > G and Y318CfsX7). According to this, counselling was conducted to advocate for oocyte donation in order to obtain a normal pregnancy. This technique enabled a complication-free, singleton pregnancy that resulted in a healthy term live birth of a 2900 g female. Few months after delivery, the patient presented a new complete hydatidiform mole. Women presented with mutations in the NLRP7, KHDC3L or PADI6 genes are unlikely to obtain normal pregnancies, with a major risk of reproductive failure. In such a context, oocyte donation may be the best option. Only 4 normal pregnancies and deliveries have been published in this situation through this technique to our knowledge.

Systematic and standardized hysteroscopic endometrial injury for treatment of recurrent implantation failure.

RESEARCH QUESTION: To investigate the effect of hysteroscopic endometrial injury for treatment of recurrent implantation failure (RIF). DESIGN: This prospective and randomized controlled trial included 239 patients who had failed to achieve a clinical pregnancy after the transfer of at least four good-quality embryos in a minimum of three fresh or frozen-thawed embryo transfer cycles and were under the age of 40 years, who were randomized into two groups. The injury group (n = 124) received endometrial injury during their hysteroscopic procedure, whereas the control group (n = 115) did not. Patients who had endometrial pathologies were excluded from the study. RESULTS: There were no statistically significant differences in duration of gonadotrophin use (8.23 versus 8.30 days), total dose of gonadotrophins (2330 versus 2338 IU), number of oocytes (7.03 versus 8.21), number of mature oocytes (5.27 versus 6.02), number of fertilized oocytes (4.19 versus 4.55), number of good-quality embryos (2.07 versus 2.43), number of embryos transferred (1.97 versus 1.93) or endometrial thickness (9.04 versus 9.35 mm) between the injury group and control group, respectively. Clinical pregnancy rates (25.8% versus 15.6%, P = 0.047), live birth rates (21.8% versus 12.2%, P = 0.049) and implantation rates (14.2% versus 8.8%, P = 0.036) were significantly different, favouring the injury group. CONCLUSION: This study suggests that endometrial injury is beneficial in RIF patients to increase the odds of implantation, clinical pregnancy and live birth.

Endometrium immunomodulation by intrauterine insemination administration of treated peripheral blood mononuclear cell prior frozen/thawed embryos in patients with repeated implantation failure.

In assisted reproductive technology (ART) programmes, approximately 10% of infertile patients have at least two or three repeated implantation failures (RIFs) after an in vitro fertilization (IVF) protocol. Successful implantation mainly depends on local immune tolerance mechanisms involving a spectrum of cytokines, interleukins and growth factors. The latter have played pivotal roles in the recruitment of immune cells (and notably T-lymphocyte cells). In total, 250 couples participating in frozen-thawed embryo transfer programme were incorporated in a randomized clinical trial (peripheral blood mononuclear cells (PBMC) subgroup: n=122; control subgroup: n=128). In the PBMC group, a blood sample was collected 5 days before the scheduled frozen-thawed embryo transfer; PBMCs were isolated using Ficoll separation and then cultured for 72 h. Two days prior to embryo transfer, 0.4 ml of cultured PBMCs were transferred into the patient's uterus. Although the clinical pregnancy rate was higher in the PBMC group (34.4%) than in the control group (23.4%), this difference was not statistically significant (P=0.05 in a chi-squared test). Nevertheless, when we limited the analysis to patients with ≥3 RIFs (n=138), there was a significant difference in the clinical pregnancy rate between the PBMC group (38.6%) and the control group (19.7%; P=0.01). Our results imply that PBMC transfer can be part of effective fertility treatment for patients with RIF.

Does the dysregulation of matrix metalloproteinases contribute to recurrent implantation failure?

INTRODUCTION: The progress in in vitro fertilization (IVF) techniques for infertility management has led to the investigation of embryo implantation site proteins such as Matrix metalloproteinases (MMPs), which may have a key role in embryo-endometrium crosstalk and in the molecular mechanisms of the embryo implantation. Areas covered: Numerous studies have generated much information concerning the relation between the different proteins at the site of implantation such as cytokines, growth factors, adhesion molecules and MMPs. However, the exact role of the MMPs in embryo implantation and the impact of their dysregulation in recurrent implantation failure have yet to be characterized. Expert commentary: The proteomic investigation of the MMPs and their molecular pathways may enable scientists and clinicians to correct this dysregulation (via appropriate means of prevention and treatment), better manage embryo transfer during IVF cycles, and thus increase the ongoing pregnancy rate.

Paternal age: Negative impact on sperm genome decays and IVF outcomes after 40 years.

This study assessed sperm quality declining on relation to paternal age and its impact on in vitro fertilization (IVF) outcomes in order to estimate the APA (Advanced Paternal Age) cutoff. For this, 83 couples undergoing IVF treatment for male factor infertility were enrolled. The women age was ≤39 years, whereas the men were divided in two groups: APA (n = 41; age ≥ 40 years) and young (Y) (n = 42; age < 40 years). Conventional semen parameters (volume, concentration, motility, vitality, and morphology) were analyzed in the collected sperm samples. Furthermore, sperm genome decays (SGD) was assessed by TUNEL assay (DNA fragmentation), aniline blue staining (chromatin decondensation), and fluorescent in situ hybridization (aneuploidy). No significant difference was found concerning the conventional semen parameters between APA and Y groups. Conversely, SGD analysis showed increased DNA fragmentation; chromatin decondensation and sperm aneuploidy rates in the APA group (respectively, 41%, 43%, and 14% vs. 25%, 23%, and 4% in Y group). IVF outcomes also were affected by paternal age as indicated by the rates of cancelled embryo transfers, clinical pregnancy and miscarriage in the two groups APA and Y (29%, 17%, and 60% vs. 10%, 32%, and 42%). Finally, statistical analysis of the results suggests that the age of 40 should be considered as the APA cutoff during ART attempts.

Genetic evaluation of patients with non-syndromic male infertility.

PURPOSE: This review provides an update on the genetics of male infertility with emphasis on the current state of research, the genetic disorders that can lead to non-syndromic male infertility, and the genetic tests available for patients. METHODS: A comprehensive review of the scientific literature referenced in PubMed was conducted using keywords related to male infertility and genetics. The search included articles with English abstracts appearing online after 2000. RESULTS: Mutations in 31 distinct genes have been identified as a cause of non-syndromic human male infertility, and the number is increasing constantly. Screening gene panels by high-throughput sequencing can be offered to patients in order to identify genes involved in various forms of human non-syndromic infertility. We propose a workflow for genetic tests which takes into account semen alterations. CONCLUSIONS: The identification and characterization of the genetic basis of male infertility have broad implications not only for understanding the cause of infertility but also in determining the prognosis, selection of treatment options, and management of couples. Genetic diagnosis is essential for the success of ART techniques and for preserving future fertility as well as the prognosis for testicular sperm extraction (TESE) and adopted therapeutics.

Exome sequencing reveals a nonsense mutation in TEX15 causing spermatogenic failure in a Turkish family.

Infertility is a global healthcare problem, and despite long years of assisted reproductive activities, a significant number of cases remain idiopathic. Our currently restricted understanding of basic mechanisms driving human gametogenesis severely limits the improvement of clinical care for infertile patients. Using exome sequencing, we identified a nonsense mutation leading to a premature stop in the TEX15 locus (c.2130T>G, p.Y710*) in a consanguineous Turkish family comprising eight siblings in which three brothers were identified as infertile. TEX15 displays testis-specific expression, maps to chromosome 8, contains four exons and encodes a 2789-amino acid protein with uncertain function. The mutation, which should lead to early translational termination at the first exon of TEX15, co-segregated with the infertility phenotype, and our data strongly suggest that it is the cause of spermatogenic defects in the family. All three affected brothers presented a phenotype reminiscent of the one observed in KO mice. Indeed, previously reported results demonstrated that disruption of the orthologous gene in mice caused a drastic reduction in testis size and meiotic arrest in the first wave of spermatogenesis in males while female KO mice were fertile. The data from our study of one Turkish family suggested that the identified mutation correlates with a decrease in sperm count over time. A diagnostic test identifying the mutation in man could provide an indication of spermatogenic failure and prompt patients to undertake sperm cryopreservation at an early age.

p,p'-DDT induces testicular oxidative stress-induced apoptosis in adult rats.

BACKGROUND: The 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (p,p'-DDT) is a known persistent organic pollutant and male reproductive toxicant. The present study is designed to test the hypothesis that oxidative stress mediates p,p'-DDT-induced apoptosis in testis. METHODS: Male Wistar rats received an intraperitoneal (ip) injection of the pesticide at doses of 50 and 100mg/kg for 10 consecutive days. The oxidative stress was evaluated by biomarkers such lipid peroxidation (LPO) and metallothioneins (MTs) levels. Antioxidant enzymes activities was assessed by determination of superoxide dismutase (SOD), catalase (CAT) and hydrogen peroxide (H2O2) production. In addition, glutathione-dependent enzymes and reducing power in testis was evaluated by glutathione peroxidase (Gpx), glutathione reductase (GR), glutathione S-transferase (GST) activities and reduced and oxidized glutathione (GSH - GSSG) levels. Apoptosis was evaluated by DNA fragmentation detected by agarose gel electrophoresis. Germinal cells apoptosis and the apoptotic index was assessed through the TUNEL assay. RESULTS: After 10 days of treatment, an increase in LPO level and H2O2 production occurred, while MTs level, SOD and CAT activities were decreased. Also, the Gpx, GR, GST, and GSH activities were decreased, whereas GSSG activity was increased. Testicular tissues of treated rats showed pronounced degradation of the DNA into oligonucleotides as seen in the typical electrophoretic DNA ladder pattern. Intense apoptosis was observed in germinal cells of DDT-exposed rats. In addition, the apoptotic index was significantly increased in testis of DDT-treated rats. CONCLUSIONS: These results clearly suggest that DDT sub-acute treatment causes oxidative stress in rat testis leading to apoptosis.

A no-stop mutation in MAGEB4 is a possible cause of rare X-linked azoospermia and oligozoospermia in a consanguineous Turkish family.

PURPOSE: The purpose of this study was to identify mutations that cause non-syndromic male infertility using whole exome sequencing of family cases. METHODS: We recruited a consanguineous Turkish family comprising nine siblings with male triplets; two of the triplets were infertile as well as one younger infertile brother. Whole exome sequencing (WES) performed on two azoospermic brothers identified a mutation in the melanoma antigen family B4 (MAGEB4) gene which was confirmed via Sanger sequencing and then screened for on control groups and unrelated infertile subjects. The effect of the mutation on messenger RNA (mRNA) and protein levels was tested after in vitro cell transfection. Structural features of MAGEB4 were predicted throughout the conserved MAGE domain. RESULTS: The novel single-base substitution (c.1041A>T) in the X-linked MAGEB4 gene was identified as a no-stop mutation. The mutation is predicted to add 24 amino acids to the C-terminus of MAGEB4. Our functional studies were unable to detect any effect either on mRNA stability, intracellular localization of the protein, or the ability to homodimerize/heterodimerize with other MAGE proteins. We thus hypothesize that these additional amino acids may affect the proper protein interactions with MAGEB4 partners. CONCLUSION: The whole exome analysis of a consanguineous Turkish family revealed MAGEB4 as a possible new X-linked cause of inherited male infertility. This study provides the first clue to the physiological function of a MAGE protein.

Involvement of oxidative stress in the mechanism of p,p'-DDT-induced nephrotoxicity in adult rats.

The 1,1,1-trichloro-2,2-bis(4-chlorophenyl) ethane (p,p'-DDT) is an organochlorine pesticide that persists in the environment and has a risk to human health. We investigated whether p,p'-DDT-induces nephrotoxicity in rats and whether oxidative stress and apoptosis are involved in the pathogenesis of this process. Male rats received the pesticide at doses of 50 and 100 mg/kg for 10 days. Renal damage was evaluated by histopathological examination and serum markers. The oxidative stress was evaluated by lipid peroxidation (LPO), metallothioneins (MTs) and protein carbonyl levels. Antioxidant enzymes were assessed by determination of superoxide dismutase (SOD) and catalase (CAT) activities. Glutathione-dependent enzymes and reducing power in kidney were evaluated by glutathione peroxidase (GPx), glutathione reductase (GR), glutathione S-transferase (GST) activities. Renal tubular cells apoptosis was assessed through the TUNEL assay. After 10 days of treatment, an increase of serum creatinine and urea levels occurred, LPO and protein carbonyl levels were increased, while MTs level, SOD and CAT activities were decreased. Besides, the GPx, GR, GST, and GSH activities were decreased. Histological alterations in kidney tissue and intense apoptosis in renal tubular cells were observed. These results suggest that DDT sub-acute treatment causes oxidative stress and apoptosis, which may be the chief mechanisms of DDT-induced nephrotoxicity.

Mechanisms of chromium hexavalent-induced apoptosis in rat testes.

Hexavalent chromium (CrVI)-containing compounds, present in industrial settings and in the environment, are known as carcinogens and mutagens. The present study is designed to test the hypothesis that oxidative stress mediates CrVI-induced apoptosis in testis. Male Wistar rats received an intraperitoneal injection of potassium dichromate at doses of 1 and 2 mg kg-1. Superoxide anion production was assessed by the determination of the reduction of cytochrome c and iodonitrotetrazolium, lipid peroxidation (LPO), metallothioneins (MTs), and catalase (CAT) activity. Apoptosis was evaluated by DNA fragmentation detected by agarose gel electrophoresis. Germinal cells apoptosis was detected by toluidine blue staining. The expression of Bax and Bcl-2 proteins (Pts) was also investigated. After 15 days of treatment, an increase of LPO and MT levels occurred, while CAT activity was decreased. Testicular tissues of treated rats showed pronounced degradation of the DNA into oligonucleotides as seen in the typical electrophoretic DNA ladder pattern. Intense apoptosis was observed in germinal cells of Cr-exposed rats. Bax Pt expression was induced in spermatogonia and spermatocytes cells of CrVI-treated rats. In contrast, Bcl-2 Pt was occasionally observed in germ cells of CrVI-exposed rats. These results clearly suggest that CrVI subacute treatment causes oxidative stress in rat testis leading to apoptosis.

Intrauterine insemination of cultured peripheral blood mononuclear cells prior to embryo transfer improves clinical outcome for patients with repeated implantation failures.

Implantation failure is a major limiting factor in assisted reproduction improvement. Dysfunction of embryo-maternal immuno-tolerance pathways may be responsible for repeated implantation failures. This fact is supported by immunotropic theory stipulating that maternal immune cells, essentially uterine CD56+ natural killer cells, are determinants of implantation success. In order to test this hypothesis, we applied endometrium immuno-modulation prior to fresh embryo transfer for patients with repeated implantation failures. Peripheral blood mononuclear cells were isolated from repeated implantation failure patients undergoing assisted reproductive technology cycles. On the day of ovulation induction, cells were isolated and then cultured for 3 days and transferred into the endometrium cavity prior to fresh embryo transfer. This immunotherapy was performed on 27 patients with repeated implantation failures and compared with another 27 patients who served as controls. Implantation and clinical pregnancy were increased significantly in the peripheral blood mononuclear cell test versus control (21.54, 44.44 vs. 8.62, 14.81%). This finding suggests a clear role for endometrium immuno-modulation and the inflammation process in implantation success. Our study showed the feasibility of intrauterine administration of autologous peripheral blood mononuclear cells as an effective therapy to improve clinical outcomes for patients with repeated implantation failures and who are undergoing in vitro fertilization cycles.