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RESEARCH QUESTION: Does magnetic-activated cell sorting (MACS) for sperm selection increase cumulative live birth rates (CLBR) or improve clinical parameters of reproductive success in couples undergoing intracytoplasmic sperm injection (ICSI) with donor oocytes? DESIGN: Retrospective multicentre observational study including data compiled from unselected couples who underwent ICSI cycles with donated oocytes in 15 Spanish IVIRMA fertility clinics (January 2008 to February 2020). Patients were divided into reference (standard semen processing, n = 40,157) and MACS (additional sperm selection step by MACS, n = 1,240) groups. CLBR were plotted on Kaplan-Meier curves and compared using the Mantel-Cox test. Proportions were compared with a generalized estimating equation model, and results were adjusted to clinically relevant variables. RESULTS: The MACS group showed a 27.1% CLBR after one embryo was transferred and 81.6% after four; the reference group had CLBR of 19.6% and 78.5%, respectively. CLBR in the MACS group was 4.2% after five oocytes were used and 75.5% after 15; for the reference group, CLBR were 7.8% and 78.3%, respectively. Kaplan-Meier curves showed statistically significant differences in CLBR per number of embryos transferred and per number of donated metaphase II oocytes between the two groups (both P

Research question: Does time since vasectomy (as obstructive interval) and the presence of different male comorbidities adversely affect the likelihood of achieving a newborn for vasectomized males undergoing testicular sperm extraction (TESE) and intracytoplasmic sperm injection (ICSI)? Design: This retrospective study included 364 couples with vasectomized males undergoing TESE-ICSI cycles with autologous oocytes at IVI Valencia. The main outcome was live birth rate (LBR). Subjects were divided according to the male risk factor evaluated into quartiles (obstructive interval, body mass index [BMI]) or groups (hypertension, diabetes mellitus, dyslipidaemia). The reproductive outcomes were calculated per embryo transfer, per ovarian stimulation completed, and per couple. Results: The average obstructive interval was 11.3 years. The LBR was 34.4% (95% CI 30.1–38.6) per embryo transfer, 27.8% (95% CI 24.1–31.5) per ovarian stimulation and 46.2% (95% CI 41.8–51.3) per couple. When considering obstructive interval, a significantly lower LBR per couple (P = 0.04) was found in the group with the longest obstruction time: Q1 42.1% (95% CI 33.5–50.7), Q2 49.1% (95% CI 36.1–62.1), Q3 56.3% (95% CI 46.7–65.9) and Q4 37.2% (95% CI 26.5–47.9) but the cumulative live birth rate (CLBR) was not affected (P = 0.63). LBR per ovarian stimulation of males with hypertension was significantly lower (P = 0.04) than healthy males: 13.5% (95% CI 2.5–24.5) and 28.6% (95% CI 24.7–32.5), respectively. The group of diabetic vasectomized males had a significantly higher CLBR (P = 0.02). The remaining risk factors assessed (smoking, dyslipidaemia and a high BMI) did not affect LBR compared with their healthy counterparts. Conclusion: Time since vasectomy appears to negatively influence the LBR when assessed per couple. The CLBR was not affected by the obstructive interval or the presence of other male comorbidities apart from diabetes, which had a significant effect.

Do donor spermatozoa improve IVF outcomes after first oocyte donation failure?Retrospective, multicentre study including couples undergoing oocyte donation cycles using autologous or donor spermatozoa after a failed first attempt. Male partners were further characterized as normozoospermic or oligoasthenoteratospermic, i.e. fewer than 5 million motile progressive spermatozoa in the ejaculate. The main outcomes measured were live birth rate (LBR) per embryo transfer, LBR per number of embryos transferred, and cumulative LBR (CLBR) considering oocytes consumed in the previous donation cycles.Analysis comprised 6065 cycles of oocyte donation failure; among these, subgroup analyses by sperm quality comprised 4113 cycles with severe male factor and 1150 cycles with suboptimal/normal spermatozoa. Sperm replacement in the first cycle after failure increased LBR per embryo transfer (OR 2.21, 95% CI 1.7-2.8, P 0.001) and per number of embryos transferred (OR 2.46, 95% CI 1.9-3.1, P 0.001) for normospermic and oligoasthenoteratospermic men. Replacement by the third cycle after failure was less beneficial (LBR per embryo transfer: OR 1.35, 95% CI 0.9-2.1, P = 0.16; LBR per embryos transferred: OR 1.33, 95% CI 0.9-2.0, P = 0.186). Kaplan-Meier curves of CLBR per oocyte fertilized with autologous or donor spermatozoa were statistically different (P 0.001) and demonstrate how each additional oocyte may affect success based on sperm source (donor/autologous).Donor spermatozoa improved outcomes when used after an initial failed oocyte donation cycle. The CLBR curves can be used to determine the cumulative chances of live birth using either autologous or donor spermatozoa, providing guidance on when to replace spermatozoa.

Study question Does an elevated SDF (>15%) increase the odds of adverse maternal and neonatal outcomes in autologous oocyte IVF-ICSI cycles from unselected couples? Summary answer No adverse effects of high SDF on obstetric and neonatal outcomes have been found in couples with sperm fragmentation undergoing IVF-ICSI cycles with own eggs. What is known already Sperm chromatin integrity assessment has been implemented as an additional tool in the clinical evaluation of sperm quality in infertile patients undergoing an assisted reproduction treatment. All of the published reports to date appraise its effect on clinical outcomes, and how it impacts embryo quality and the pregnancy chances after IVF and ICSI cycles. Sperm DNA integrity has also been hypothesized to affect offspring health but not many studies have reported in humans if an elevated SDF raises the risks of obstetric, delivery and neonatal outcomes. Study design, size, duration Multicentric retrospective cohort study of all IVF-ICSI cycles using autologous oocytes between January 2000-March 2019 at Spain IVIRMA clinics of couples with a SDF test on their ejaculated semen. The sperm fragmentation index was measured in all men with TUNEL assay. The database included 228 couples which had a delivery with at least a newborn. Subjects were divided into two study groups according to their level of SDF: ≤15% (low SDF) or > 15% (high SDF). Participants/materials, setting, methods Patients with missed information on maternal and neonatal outcomes were not counted for the analysis. The obstetric outcomes were gestational age, gestational diabetes, preeclampsia (hypertension with proteinuria after 20 weeks of gestation) and type of delivery. Neonatal outcomes were sex, birth weight, length, head circumference, Apgar score at 1, 5, 10 minutes, and neonatal intensive care unit (NICU) admission. Student’s t-test and Fisher’s test were used for statistical analysis. A p-value Main results and the role of chance Maternal age mean was 37.4 years (95%CI 36.9-38.0) in ≤ 15%SDF group and 37.2 years (95%CI 36.1-38.4) in > 15%SDF group (p = 0.8). Similar gestational age was found, 41.8 weeks (95%CI 41.3-42.2) in ≤ 15%SDF and 41.3 weeks (95%CI 40.4-42.3) in > 15%SDF. Gestational diabetes incidence was higher in > 15%SDF compared to ≤ 15%SDF group (3.5% versus 1.7% (OR = 2.0 (95%CI 0.03-39.8), p = 0.5). Equally, the incidence of preeclampsia was 3.6% in patients with high SDF versus 1.7% in couples with low SDF, OR = 2.1 (95%CI 0.03-41.3), p = 0.5. Type of delivery frequency was in the ≤15%SDF group 61.9% vaginal and 38.1% cesarean, while in the >15%SDF group 62.1% vaginal and 37.9% cesarean (OR = 1.0 (95%CI 0.4-2.6), p = 1.0). The overall proportion of singleton pregnancies was 87.2% (95%CI 82.4-91.2) and twins 12.8% (95%CI 8.8-17.6). There were no statistically differences between groups in the rate of delivery of twins and in the sex ratio of the newborns. When comparing the newborns of ≤ 15%SDF with >15%SDF group, the average of weight was 3011.7g (95%CI 2912.2-3111.2) versus 2986.4g (95%CI 2753.1-3219.7), of length was 49.2cm (95%CI 48.3-50.0) versus 49.5cm (95%CI 49.2-49.9), of head circumference was 34.9cm (95%CI 34.6-35.2) versus 34.3cm (95%CI 33.4-35.2). No statistically differences were observed for Apgar punctuation and for NICU admission. Limitations, reasons for caution Due to the retrospective nature of the study we have missing data from the lack of follow-up of many patients after the confirmation of the ongoing pregnancy. Although pregnancies of couples with elevated SDF have a higher incidence of gestational diabetes and preeclampsia, the sample size evaluated is a limitation. Wider implications of the findings This is one of the first reports to evaluate the relationship between paternal DNA damage and obstetric risks and neonatal health in couples with high SDF who underwent IVF-ICSI in our centers. Despite SDF did not jeopardize the maternal and neonatal outcomes, more studies are needed to confirm this conclusion. Trial registration number NA

Study question Is hyperspectral imaging of individual sperm cells an appropriate candidate technique with potential to identify molecular characteristics of single spermatozoa prior to microinjection? Summary answer Preliminary tests have allowed us to obtain the images, set up the methodology and the correct segmentation of each sperm into its signature spectra. What is known already Although image techniques such as fluorescence microscopy and Raman spectroscopy have been used to identify biomarkers on sperm cells, their translation to the fertility clinic is highly complex and unfeasible. Hyperspectroscopy has not yet been introduced in the reproductive field, but it has been successfully used in other medical disciplines to differentiate cell types or pathological versus healthy tissue. It relies on the combination of information from optical images and electromagnetic spectra produced by cells at different wavelengths. Therefore, it is an interesting candidate for objective non-destructive sperm selection before ICSI according to the presence of specific biomarkers. Study design, size, duration Pilot, prospective, observational study in the IVIRMA Valencia fertility clinic, designed as a proof of concept. Discarded semen samples were used for the technical setup and the preliminary tests of image acquisition. Hyperspectral images were obtained of 12 spermatozoa: two sperm cells were imaged 10 times each, while the other 10 sperm were imaged one time each. Participants/materials, setting, methods Samples were prepared on glass-bottom ICSI plates in PVP droplets according to routine clinical practice. Images were obtained using the camera HinaLea 4200M coupled to the Olympus IX73 inverted microscope. Hypercubes were extracted using MATLAB 2016a, and the most informative wavelength was chosen. Matrox Copilot software was used to process images and select the regions of interest (ROI) equivalent to one sperm. MATLAB was used to obtain the information for the pixels within each ROI. Main results and the role of chance Hypercube size for each image was 608x968x299 pixels. Each hypercube obtained provided information for 299 different wavelengths, from 402.8 to 998.3 nm. Once the hypercubes were obtained from each image, the spectra at 520 nm wavelength was selected as the one at which the pixel provided the most information. The background of each uploaded image was successfully removed by creating a mask, which was then used to extract and characterize all the spectra from each pixel of each sperm image. The result of this proof of concept, though measure qualitatively, is that the information obtained from hyperspectral imaging of individual sperm is highly reproducible and successful in showing overlapping spectra resulting from 10 different images of the same specimen. Spectra resulting from images of different sperm cells showed tangible differences on their composition demonstrating unique biochemical features of each. The next step will be to quantitatively measure the reproducibility and sensitivity of the technique in a larger sample, to then study the potential of single sperm hyperspectral imaging as a sperm selection tool for ICSI. Limitations, reasons for caution These are preliminary results obtained from the development of an appropriate methodology to adapt the acquisition of hyperspectral images in the clinic’s IVF laboratory. Thus, the group will continue to evaluate the reproducibility, sensitivity, and informative capability of the technique before contemplating its use on samples destined to perform cycles. Wider implications of the findings: Having developed a laboratory and image processing methodology that allows us to successfully segment each obtained image according to the hyperspectral information of every pixel of each individual sperm, we could move forward in testing the potential of these hyperspectral signatures to identify sperm markers related to successful reproductive outcomes. Trial registration number Not applicable

Personalized medicine gathers the most relevant data involved in human health. Currently, the diagnosis of male infertility is limited to spermiogram, which does not provide information on the male fertile potential. New diagnostic methods are required. The application of omics techniques in the study of male reproductive health renders a huge amount of data providing numerous novel infertility biomarkers, from genes to metabolites, to diagnose the cause of male infertility. Recent studies hold the promise that these biomarkers will allow a noninvasive infertility diagnosis and the improvement of the sperm selection techniques.