IMSI—Guidelines for Sperm Quality Assessment

Intracytoplasmic sperm injection (ICSI) is a widely used and accepted treatment of choice for oocyte fertilization. However, the quality of sperm selection depends on the accurate visualization of the morphology, which can be achieved with a high image resolution. We aim to correct the conviction, shown in a myriad of publications, that an ultra-high magnification in the range of 6000×–10,000× can be achieved with an optical microscope. The goal of observing sperm under the microscope is not to simply get a larger image, but rather to obtain more detail—therefore, we indicate that the optical system’s resolution is what should be primarily considered. We provide specific microscope system setup recommendations sufficient for most clinical cases that are based on our experience showing that the optical resolution of 0.5 μm allows appropriate visualization of sperm defects. Last but not least, we suggest that mixed research results regarding the clinical value of IMSI, comparing to ICSI, can stem from a lack of standardization of microscopy techniques used for both ICSI and IMSI.

P–238 Impaired oocyte fertilization is associated with a proinflammatory M1 phenotype of macrophages and the activation of the NLRC4 inflammasome

Study question Are inflammatory signatures in the blood of clinically asymptomatic patients predictive for oocyte fertilization success in ART treatment? Summary answer The proinflammatory M1 phenotype of macrophages combined with increasing numbers of cytotoxic T-cells and an upregulated inflammasome NLRC4 is associated with impaired oocyte fertilization. What is known already Oocyte fertilization is an indispensable step towards embryogenesis and reproductive success. Patients with acute or chronic inflammation are at risk of reduced fertilization rates and failure of assisted reproductive technology (ART) treatment. Inflammasomes are intracellular multiprotein complexes that mediate inflammatory tissue remodeling. They can be activated by endogenous and exogenous stimuli and result in maturation of the cytokines IL–1βand IL–18. Appropriate inflammasome activation serves for pathogen defense and tissue damage repair, while aberrant activation of inflammasomes can enhance uncontrolled tissue damage. Study design, size, duration This a prospective study including 39 patients stratified by fertilization rate (cut off 66%) of mature M II oocytes after ICSI procedure, compares 20 patients with high (mean 84,8%, range 67 –100%) versus 19 patients with low (mean 29.4%, range 0–65%) rates. Participants/materials, setting, methods We performed FACS analysis of immune cell composition (leukocytes, neutrophils, monocytes, macrophage types M1 and M2, cytotoxic T-cells, T-helper cells) and utilized RT-qPCR analysis of three inflammasomes (AIM 2, NLRP3 and NLRC4) and their down-streaming proteins caspase 1 and IL–1ß. We focused on an initial and a late state during controlled ovarian stimulation (COS) procedure. Main results and the role of chance This study reports a cellular and molecular inflammatory signature associated with reduced oocyte fertilization rates after ICSI treatment in clinically asymptomatic patients. On a cellular level proinflammatory M1 macrophages were significantly elevated in the low fertilization group (p < 0.01), additionally the ratio of proinflammatory M1 macrophages to anti-inflammatory M2 phenotype was inversely associated with oocyte fertilization rate (p < 0.001). Cytotoxic T cells (p < 0.05) and the inflammasome NLRC4 (p < 0.01) revealed identical patterns of association with fertilization rates in the group comparison. In line with this pattern, there was a significant upregulation of Caspase 1 with a consecutive increase in IL–1ß activity in women with low fertilization rates (p < 0.05). No significant association was shown for the other tested immune cells (leukocytes, neutrophils, monocytes, T-helper cells) and inflammasomes (NLRC3, AIM2). Limitations, reasons for caution The preliminary results in this small study indicate an activation of the inflammasome NLRC4. This, however, has to be assessed on the protein level in a larger group of patients. Wider implications of the findings: Systemic macrophage augmentation of M1 phenotype and cytotoxic T cell presence in combination with upregulated inflammasome NLRC4 are associated with impaired oocyte fertilization. The predictive information of the identified immunologic signature could indicate targeted treatment to down regulate systemic inflammation and the use of oocyte activators in the ART laboratory. Trial registration number none

Sperm modulate uterine immune parameters relevant to embryo implantation and reproductive success in mice

Seminal fluid factors modulate the female immune response at conception to facilitate embryo implantation and reproductive success. Whether sperm affect this response has not been clear. We evaluated global gene expression by microarray in the mouse uterus after mating with intact or vasectomized males. Intact males induced greater changes in gene transcription, prominently affecting pro-inflammatory cytokine and immune regulatory genes, with TLR4 signaling identified as a top-ranked upstream driver. Recruitment of neutrophils and expansion of peripheral regulatory T cells were elevated by seminal fluid of intact males. In vitro, epididymal sperm induced IL6, CXCL2, and CSF3 in uterine epithelial cells of wild-type, but not Tlr4 null females. Collectively these experiments show that sperm assist in promoting female immune tolerance by eliciting uterine cytokine expression through TLR4-dependent signaling. The findings indicate a biological role for sperm beyond oocyte fertilization, in modulating immune mechanisms involved in female control of reproductive investment.

Fertilization by ICSI generates a higher number of live births than IVF in a pioneer facility applying >90% single blastocyst-stage embryo transfers

Following earlier studies introducing an IVF-ICSI Split model on couples with unexplained infertility to avoid the scenario of unexplained failed or poor fertilization, PIVET has adopted a high ICSI rate approaching 90%, whereas the general rate among Australian facilities is around 60%. This observational study with retrospective data analysis reports on the IVF±ICSI procedures conducted over the period 2011 to 2019 with follow-up of ensuing pregnancies through 2020. Using autologous oocytes, 2343 women had 3434 IVF±ICSI cycles where 84.5% of women had 88.9% of initiated treatment cycles using ICSI and only 5.3% of women had 4.0% of cycles by IVF. The remaining 10.1% of women utilized the IVF-ICSI Split model for the remaining 7.2% of cycles. It was shown that oocyte fertilization rates were significantly higher for ICSI (p<0.0001), but not significant for women >40 years. The utilization rates of the ensuing embryos were ~45% across all ages with no significant differences across the ages, except for those small numbers of women ≥45 years who had a higher rate from IVF-generated embryos (p<0.0002). Pregnancy outcome were higher from ICSI-generated embryos across the age groups, being especially marked among the younger women <40 years (p<0.0001). Miscarriage rates were lowest for the IVF-generated pregnancies (overall 6.7% vs 22.8%, p<0.0001) but nevertheless the final live birth productivity rates per initiated treatment cycle remained higher from the ICSI-generated pregnancies (56.5% vs 46.3%; p<0.0001). Although this study does not meet the highest standards for EBM, it emanates from a pioneer facility with >40 years of published activity and which practices 90% blastocyst transfers in >90% SET cycles. The study supports a high ICSI rate of almost 90% and an IVF-ICSI Split rate of 10%.

Eclampsia-Induced Posterior Reversible Encephalopathy Syndrome in a Donor Oocyte Recipient

Objective: Posterior reversible encephalopathy syndrome (PRES) has been robustly associated with preeclampsia, hyperperfusion or endothelial dysfunction suggested as possible mechanisms. In this article, we report an illustrative case of this complication in a patient with risk factors for hypertensive disorders in pregnancy, including advanced maternal age and donor oocyte fertilization. Case report: We present a case of a 40-year-old pregnant, donor oocyte recipient with sudden decreased visual acuity accompanied by hypertension, proteinuria and tonic-clonic seizures. Magnetic resonance imaging (MRI) of the brain showed bilateral lesions in the parieto-occipital regions suggestive of vasogenic edema, leading us to suspect posterior reversible encephalopathy syndrome. The patient underwent an emergency cesarean section and labetalol and magnesium sulfate were administered intravenously. The neurological symptoms and radiological findings resolved following delivery and the patient’s blood pressure normalized, supporting the diagnosis of posterior reversible encephalopathy syndrome. Conclusion: Pregnancy by donor oocyte fertilization may entail a higher risk of eclampsia and associated posterior reversible encephalopathy syndrome.

Inseminating dose for the artificial fertilization of Brycon amazonicus (Teleostei: Characidae)

Summary Knowledge of the sperm–oocyte ratio in fish fertilization serves as the basis for studies on artificial reproduction and gamete manipulation. The aim of this study was to determine the minimum insemination dose for Brycon amazonicus oocyte fertilization. Female and male gametes were used and tested with the following doses of spermatozoa oocyte–1 ml–1: 10,000, 20,000, 40,000, 60,000 and 80,000 (in triplicate). Fertilization rates were calculated and estimated from the regression equation by applying the segmented regression model ‘Linear Response Plateau’ to determine the appropriate proportion of gametes. Based on the equation Ŷ = 14.3415 + 0.0007836X, the fertilization rate increased up to 63.34% as it reached a plateau with a proportion of 62,524 spermatozoa oocyte–1 ml–1, which is the minimum insemination dose recommended for artificial insemination of the species.