First-line infertility treatment in normal or subnormal sperm: Interest of a simplified pre-IMSI test.

BACKGROUND: In the field of male infertility, when sperm is normal/subnormal, a few "add-on" routine tests can complete the basic semen examination. OBJECTIVES: The aim of this study was to develop and evaluate a faster, simplified motile sperm organelle morphology examination (MSOME) technique for selected infertile patients with apparently normal/subnormal sperm and, in their background: failure of two or three intrauterine insemination (IUI) cycles, repeatedly fragmented embryos, embryonic development to blastocyst-stage failures, repeated miscarriages, a long period of infertility or 2 or more IVF attempts without pregnancy. Our test results were correlated with IUI, conventional in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI) and intracytoplasmic morphologically selected sperm injection (IMSI) outcomes. MATERIALS AND METHODS: We validated an adapted version of the MSOME analysis called the pre-IMSI test (PIT), based on vacuole evaluation alone. 248 infertile patients from our assisted reproductive technology (ART) Center were retrospectively selected and split into three PIT score subgroups (patients with ≤8% (score I), 9 to 15% (score II) and ≥16% normal spermatozoa (score III)) based on the correlation between PIT results and each ART technique outcome. The choice of one or another of these ART techniques had been made according to the usual clinico-biological criteria. RESULTS: Clinical outcomes for each of the three PIT subgroups were compared individually for the different ART techniques. For ICSI, the effect of the PIT score subgroup was significant for clinical pregnancies (p = 0.0054) and presented a trend for live births (p = 0.0614). Miscarriage rates of IVF attempts were statistically different depending on the PIT score (p = 0.0348). Furthermore, the odds ratios of clinical pregnancy rates were significantly different according to PIT score subgroup when comparing ICSI vs. IMSI or IVF vs. ICSI attempts. DISCUSSION: IMSI appears to be recommended when sperm belongs to PIT score I, ICSI when it belongs to PIT score II and IVF or IUI when sperm is of PIT score III quality in selected infertile couples. The lack of statistical power in these PIT subgroups means that we must remain cautious in interpreting results. CONCLUSION: Our results support the interest of this simplified test for certain couples with normal/subnormal sperm to help choose the most efficient ART technique, even as first-line treatment.

Impact of Caesarean section on pregnancy outcomes in ART after transfer of one or more frozen blastocysts.

INTRODUCTION: The prevalence of Caesarean delivery is rising steadily worldwide, and it is important to identify its future impact on fertility. A number of articles have been published on this subject, but the impact of Caesarean section on reproductive outcomes is still under debate, and none of these articles focus exclusively on frozen blastocysts. OBJECTIVE: The aim of this study was to evaluate the impact of a previous Caesarean delivery compared with a previous vaginal delivery on the chances of a live birth following the transfer of one or more frozen embryos at the blastocyst stage. METHODS: This was a retrospective, bicentric study at the University Hospitals of Nîmes and Montpellier, conducted between January 1st, 2016 and February 1st, 2021. Three hundred and ninety women with a history of childbirth and a transfer of one or more frozen embryos at blastocyst stage were included in the analysis. The primary outcome was the number of live births. Secondary outcomes were: the rate of positive HCG, miscarriage, ectopic pregnancy and clinical pregnancy, as well as the live birth rate according to the presence or absence of an isthmocele. RESULTS: Of the 390 patients included, 118 had a previous Caesarean delivery and 272 a vaginal delivery. No statistically significant differences were found for the primary (p = 0.9) or secondary outcomes. A trend towards lower live birth rates was observed in patients with isthmoceles, but this did not reach significance (p>0.9). On the other hand, transfers were more often described as difficult in the Caesarean delivery group (p = 0.011). CONCLUSION: Our study found no effect of previous Caesarean delivery on the chances of live birth after transferring one or more frozen blastocysts. However, further prospective studies are needed to confirm these results.

Fertility outcome after Intracytoplasmic Sperm Injection with surgically retrieved sperm.

BACKGROUND: Surgically retrieved sperm samples and Intra Cytoplasmic Sperm Injection are indicated when we face severe sperm abnormalities such as azoospermia. The objective of this study was to assess the rates of clinical pregnancies and live births with sperm from testicular biopsies. DESIGN: This was a retrospective descriptive study of all Intra Cytoplasmic Sperm Injection (ICSI) cycles performed with surgically retrieved sperm at the Nîmes University Hospital from January 1st, 2015 to December 31st, 2018. The main outcome was the clinical pregnancy rate. The secondary outcomes were the fertilization rate, the implantation rate and the live birth rate. RESULTS: In all, 99 couples were treated at our center during this period and 164 ICSI cycles using surgically-collected sperm were performed. The men were 34.7 ± 7.4 years old on average at the time of undergoing testicular biopsy and the women were 32.15 ± 5.07 years at the time of ICSI. Out of 127 fresh embryo transfers, we observed 47 clinical pregnancies (37 %) and 45 live births (35.4 %). The average fertilization rate was 45 % and the implantation rate was 23.7 %. Among these infertile couples, 53 % obtained at least one clinical pregnancy and 42 % at least one live birth. CONCLUSION: Testicular sperm extraction combined with ICSI is indicated in cases of male infertility linked with azoospermia or severe semen alteration. This technique offers couples a marvelous opportunity to have children with their own gametes.

Interspecies differences in the cytochrome P450 activity of hepatocytes exposed to PLGA and silica nanoparticles: an in vitro and in vivo investigation.

Nanomedicines represent a promising approach in the treatment and diagnosis of numerous disorders. The majority of the injected dose of nanoparticles (NPs) is sequestrated in the liver. Despite this hepatic tropism, the interaction of NPs with the detoxification function of the liver remains unclear. The present study consists of evaluating the impact of biodegradable poly(lactide-co-glycolide) (PLGA) and silica NPs on cytochrome P450 (CYP) activities. The effects of NPs were evaluated in vitro on human and rat hepatocytes in primary cultures and in vivo by intravenous injections in healthy rats. More than the physicochemical properties, the composition of NPs (organic, inorganic) dramatically influenced the detoxification function of the liver. Silica NPs modulated the CYP activity both in rat and human hepatocytes, in contrast to PLGA NPs. A CYP isoform-dependent effect was reported and the modulation of the metabolic hepatic activity was species-dependent. Human hepatocytes were sensitive to an exposure to PLGA NPs, whereas no marked effect was detected in rat hepatocytes. The in vitro data obtained in rat hepatocytes were correlated with the in vivo data. This study emphasizes the interest to set up relevant in vitro models using human hepatic cells to evaluate the hepatotoxicity of nanomedicines.

Intracytoplasmic morphologically selected sperm injection (IMSI) after repeated IVF or ICSI failures: a prospective comparative study.

OBJECTIVE: Sperm morphology plays a significant role in assisted reproductive technologies and is associated with high implantation rates. The objective of this study was to evaluate the outcome of intracytoplasmic morphologically selected sperm injection (IMSI) after repeated failures of conventional in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) techniques. STUDY DESIGN: In a prospective study in which couples acted as their own controls, 75 infertile couples were offered IMSI after at least two previous IVF or ICSI failures. The main outcome measures were embryo quality and number of blastocysts obtained. RESULTS: The percentage of top quality embryos obtained at day 2 was increased in IMSI compared to IVF/ICSI cycles (89.8% versus 79.8%; p=0.009). Extended embryo culture was possible in 41.3% of IMSI cycles versus 26.7% of IVF/ICSI cycles (p=0.04), and the mean number of blastocysts obtained was higher in IMSI cycles (1.5±1.9) than in IVF/ICSI cycles (1.0±1.2) (p=0.03). Moreover, IMSI resulted in clinical pregnancy and birth rates of respectively 29.3% and 18.6%. CONCLUSION: After two or three IVF/ICSI failures, IMSI seems to give better embryo quality and more blastocysts, which allow more embryo transfers at the blastocyst stage. This study supports the use of sperm ultramorphology examination as an independent test to be proposed after repeated IVF or ICSI failures.

Advantages of the two-step embryo transfer strategy in human IVF/ICSI cycles.

The aim of this study was to evaluate the advantages of the two-step embryo transfer (ET) strategy combining a day 2/3 ET with a day 5/6 blastocyst transfer. In an observational comparative study, 400 infertile women were enrolled from two assisted reproductive technology (ART) units according to inclusion criteria: age below 42 years and at least three embryos obtained on day 2 thus allowing an extended in vitro culture. Two groups were defined according to the ET strategy adopted: group 1 had a two-step ET; and group 2 had a day 2/3 ET with (subgroup 2a) or without (subgroup 2b) blastocysts cryopreserved on day 5/6. Live birth rate was significantly higher in group 1 than in subgroups 2a and 2b (36.5% versus 29.4% and 13.4%, respectively; p < 10(-3)). Multiple pregnancy rates were comparable between groups. After adjusting on major prognostic factors, the two-step ET strategy was still associated with a significantly higher live birth rate than the day 2/3 ET (OR = 2.23; 95% CI: 1.32-3.77). The two-step ET provides better live birth rates than the cleavage-stage ET. It does not increase multiple pregnancy rates if the number of embryos transferred is limited. It also prevents cycle loss when embryos fail to develop into blastocysts.

Adverse effects of hepatitis B virus on sperm motility and fertilization ability during IVF.

The consequences of hepatitis B virus (HBV) infection for fertility are still unclear. Spermatozoa with decreased motility have been reported in HBV-infected patients. It has been demonstrated in vitro that HBV S protein has adverse effects on human sperm function with consequences for fertilization. In a case-control study design, 32 IVF cycles in couples with male HBV infection were compared with 64 cycles in non-infected couples, matched for age, time period, cycle rank and sperm parameters on the day of oocyte retrieval. Sperm motility before selection was significantly reduced in the HBV group (36.3 ± 11.6% versus 45.3 ± 14.4%,P = 0.003). A low fertilization rate (LFR) was more frequently observed in the HBV group (34.4% versus 15.6%, P = 0.036) and was associated with a decreased number of embryos available for transfer, although embryo quality on day 2 or 3 was not different.Implantation and pregnancy rates were comparable between groups. This study shows that HBV has a deleterious effect on sperm motility in vivo and that couples whose male partner is infected have a higher risk of LFR after IVF, a risk which is independent from the initial sperm motility.

Impact of freezing/thawing technique on sperm DNA integrity in HIV-1 patients.

INTRODUCTION: According french legislation, sperm freezing/thawing procedures are used to prevent ART contaminations in couple with HIV-1 infected men. We determined sperm nuclear fragmentation rate before and after selection and freezing/thawing in HIV-1 14 patients. METHODS: Two groups of patients were studied: 20 control patients with normal sperm (group 1) and without viral infection and 20 fertile treated HIV-1 patients (group 2). DNA fragmentation was evaluated using terminal uridine nick end labeling, before and after gradient selection, and after cryopreservation and thawing procedures. RESULTS: DNA fragmentation rates in fresh semen were increased in HIV patients (6.38% vs 3.39%) (p < 0.05) compared with control patients. After sperm migration, fragmentation rates were significantly lower (p < 0.0001) in the two groups compared with fresh sperm rates. After freezing/thawing, values were similar to those of fresh semen with an increased rate (p < 0.01) for HIV-1 patients, with respectively 3.40% and 5.18% rates in control and infected patients. HIV-1-infected patients treated by antiretroviral therapy showed a significant increase in sperm DNA fragmentation in fresh sperm and also after freezing/thawing procedures, but these two fragmentation rates were not significantly different. CONCLUSION: So, freezing/thawing procedures do not seem to impair sperm DNA and preserve probability of conception for couples with HIV-1 infected men.

Matrix metalloproteinases contribute distinct roles in neuroendocrine prostate carcinogenesis, metastasis, and angiogenesis progression.

Prostate cancer is the leading form of cancer in men. Prostate tumors often contain neuroendocrine differentiation, which correlates with androgen-independent progression and poor prognosis. Matrix metalloproteinases (MMP), a family of enzymes that remodel the microenvironment, are associated with tumorigenesis and metastasis. To evaluate MMPs during metastatic prostatic neuroendocrine cancer development, we used transgenic mice expressing SV40 large T antigen in their prostatic neuroendocrine cells, under the control of transcriptional regulatory elements from the mouse cryptdin-2 gene (CR2-TAg). These mice have a stereotypical pattern of tumorigenesis and metastasis. MMP-2, MMP-7, and MMP-9 activities increased concurrently with the transition to invasive metastatic carcinoma, but they were expressed in different prostatic cell types: stromal, luminal epithelium, and macrophages, respectively. CR2-TAg mice treated with AG3340/Prinomastat, an MMP inhibitor that blocks activity of MMP-2, MMP-9, MMP-13, and MMP-14, had reduced tumor burden. CR2-TAg animals were crossed to mice homozygous for null alleles of MMP-2, MMP-7, or MMP-9 genes. At 24 weeks CR2-TAg; MMP-2(-/-) mice showed reduced tumor burden, prolonged survival, decreased lung metastasis, and decreased blood vessel density, whereas deficiencies in MMP-7 or MMP-9 did not influence tumor growth or survival. Mice deficient for MMP-7 had reduced endothelial area coverage and decreased vessel size, and mice lacking MMP-9 had increased numbers of invasive foci and increased perivascular invasion, as well as decreased tumor blood vessel size. Together, these results suggest distinct contributions by MMPs to the progression of aggressive prostate tumor and to helping tumors cleverly find alternative routes to malignant progression.

Rat Merkel cells are mechanoreceptors and osmoreceptors.

Merkel cells (MCs) associated with nerve terminals constitute MC-neurite complexes, which are involved in slowly-adapting type I mechanoreception. Although MCs are known to express voltage-gated Ca2+ channels and hypotonic-induced membrane deformation is known to lead to Ca2+ transients, whether MCs initiate mechanotransduction is currently unknown. To answer to this question, rat MCs were transfected with a reporter vector, which enabled their identification.Their properties were investigated through electrophysiological studies. Voltage-gated K+, Ca2+ and Ca2+-activated K+ (KCa)channels were identified, as previously described. Here, we also report the activation of Ca2+ channels by histamine and their inhibition by acetylcholine. As a major finding, we demonstrated that direct mechanical stimulations induced strong inward Ca2+ currents in MCs. Depolarizations were dependent on the strength and the length of the stimulation. Moreover, touch-evoked currents were inhibited by the stretch channel antagonist gadolinium. These data confirm the mechanotransduction capabilities of MCs. Furthermore, we found that activation of the osmoreceptor TRPV4 in FM1-43-labeled MCs provoked neurosecretory granule exocytosis. Since FM1-43 blocks mechanosensory channels, this suggests that hypo-osmolarity activates MCs in the absence of mechanotransduction. Thus, mechanotransduction and osmoreception are likely distinct pathways.

Merkel cells as putative regulatory cells in skin disorders: an in vitro study.

Merkel cells (MCs) are involved in mechanoreception, but several lines of evidence suggest that they may also participate in skin disorders through the release of neuropeptides and hormones. In addition, MC hyperplasias have been reported in inflammatory skin diseases. However, neither proliferation nor reactions to the epidermal environment have been demonstrated. We established a culture model enriched in swine MCs to analyze their proliferative capability and to discover MC survival factors and modulators of MC neuroendocrine properties. In culture, MCs reacted to bFGF by extending outgrowths. Conversely, neurotrophins failed to induce cell spreading, suggesting that they do not act as a growth factor for MCs. For the first time, we provide evidence of proliferation in culture through Ki-67 immunoreactivity. We also found that MCs reacted to histamine or activation of the proton gated/osmoreceptor TRPV4 by releasing vasoactive intestinal peptide (VIP). Since VIP is involved in many pathophysiological processes, its release suggests a putative regulatory role for MCs in skin disorders. Moreover, in contrast to mechanotransduction, neuropeptide exocytosis was Ca(2+)-independent, as inhibition of Ca(2+) channels or culture in the absence of Ca(2+) failed to decrease the amount of VIP released. We conclude that neuropeptide release and neurotransmitter exocytosis may be two distinct pathways that are differentially regulated.

Hepatic environment elicits monocyte differentiation into a dendritic cell subset directing Th2 response.

BACKGROUND/AIMS: Dendritic cells (DCs), which play a critical role during immune response, could present alternative differentiation patterns depending on tissue microenvironment. Our aim was to examine the influence of hepatic microenvironment on human monocyte differentiation into DCs. METHODS: Cytology, immunophenotyping, cytokine production and T-cell activation were analyzed in DCs differentiated from human monocytes co-cultured with rat liver epithelial cells (RLEC) or human cells from various tissue origins and compared to control DCs obtained on plastic with GM-CSF/IL-4. RESULTS: RLEC environment promotes DC differentiation in the presence of IL-4 without GM-CSF. These DCs evidence similar expression of MHC-II, co-stimulatory and adhesion molecules than control DCs, but distinct lineage markers defining a CD11c+/CD14+/CD123+ DC subset. This phenotype is common to DCs from RLEC and human liver environment and differs from that obtained with skin or intestine environments. Functionally, they produce IL-10 but not IL-12p70 and favor IL-4/IL-10 secretion by T-cells rather than IFN-gamma. CONCLUSIONS: Our results confirm that tissue niches modulate DC differentiation and demonstrate that hepatic environment influences monocyte differentiation into a DC subset directing Th2 response, a key data for understanding the specialized immune response in liver. They also make RLEC co-culture system useful for studying liver DC functions.

Mice deficient in Ext2 lack heparan sulfate and develop exostoses.

Hereditary multiple exostoses (HME) is a genetically heterogeneous human disease characterized by the development of bony outgrowths near the ends of long bones. HME results from mutations in EXT1 and EXT2, genes that encode glycosyltransferases that synthesize heparan sulfate chains. To study the relationship of the disease to mutations in these genes, we generated Ext2-null mice by gene targeting. Homozygous mutant embryos developed normally until embryonic day 6.0, when they became growth arrested and failed to gastrulate, pointing to the early essential role for heparan sulfate in developing embryos. Heterozygotes had a normal lifespan and were fertile; however, analysis of their skeletons showed that about one-third of the animals formed one or more ectopic bone growths (exostoses). Significantly, all of the mice showed multiple abnormalities in cartilage differentiation, including disorganization of chondrocytes in long bones and premature hypertrophy in costochondral cartilage. These changes were not attributable to a defect in hedgehog signaling, suggesting that they arise from deficiencies in other heparan sulfate-dependent pathways. The finding that haploinsufficiency triggers abnormal cartilage differentiation gives insight into the complex molecular mechanisms underlying the development of exostoses.

Simultaneous measurement of 23 isoforms from the human cytochrome P450 families 1 to 3 by quantitative reverse transcriptase-polymerase chain reaction.

Drug metabolism in humans is essentially performed by three cytochrome P450 (P450) families (1 to 3), including 23 isoforms. The expression of these P450s is highly variable, and the rate and nature of the metabolites produced depend on the nature and the concentration of individual isoforms. P450 expression pattern is therefore a necessary tool to evaluate the effects of a given drug on P450 expression, its potential toxicity, and eventual interference with other drugs administered concomitantly. This pattern provides a general outline of the induction/repression effects of drugs leading to further mechanistic studies. A real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) assay was developed to evaluate the overall P450 expression pattern and measure human CYP1 to CYP3 mRNAs involved in drug metabolism. Our RT-PCR-based P450 mRNA assay enables us to quantify P450s rapidly with high specificity, a single annealing temperature, and low amounts of biological sample. All 23 single assays were validated by assessing the effects (induction or repression) of known inducers (ethanol, 3-methylcholanthrene, rifampicin, dexamethasone, phenobarbital) on P450 expression in human primary hepatocytes. Since this method may be used to determine human P450 expression in any human tissue or cell culture, it is a valuable tool for reliable prediction of drug safety, drug toxicity, and drug-drug interference.

Gingival organotypic culture and langerhans cells: a tool for immunotoxicologic experiments.

Langerhans cells (LCs) are dendritic cells localized in epidermis and mucosal tissues, where they are responsible for triggering the immune response. To study LCs in the oral epithelium, organotypic cultures were prepared using gingival explants. Immunochemical techniques using anti-CD1a, anti-HLA-DR, and anti-Langerin antibodies were used to detect and quantify LCs at various times. Observations were made by light and confocal microscopy. Quantification studies showed that there is a statistically significant drop in LC numbers in the epithelial tissue after 96 h of incubation. Gingival organotypic cultures thus are a good model for studying the migration of LCs and their involvement in contact hypersensitivity and periodontal diseases. The model offers potential utility as a tool for the study of periodontal tissue in the presence of different stimuli and for conducting immunotoxicologic experiments.

Cyclins E1 and E2 are required for endoreplication in placental trophoblast giant cells.

In mammalian cells, cyclin E-CDK2 complexes are activated in the late G1 phase of the cell cycle and are believed to have an essential role in promoting S-phase entry. We have targeted the murine genes CCNE1 and CCNE2, encoding cyclins E1 and E2. Whereas single knockout mice were viable, double knockout embryos died around midgestation. Strikingly, however, these embryos showed no overt defects in cell proliferation. Instead, we observed developmental phenotypes consistent with placental dysfunction. Mutant placentas had an overall normal structure, but the nuclei of trophoblast giant cells, which normally undergo endoreplication and reach elevated ploidies, showed a marked reduction in DNA content. We derived trophoblast stem cells from double knockout E3.5 blastocysts. These cells retained the ability to differentiate into giant cells in vitro, but were unable to undergo multiple rounds of DNA synthesis, demonstrating that the lack of endoreplication was a cell-autonomous defect. Thus, during embryonic development, the needs for E-type cyclins can be overcome in mitotic cycles but not in endoreplicating cells.