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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.

The use of testicular sperm is confined to patients with azoospermia, but there is evidence to support its use in males with poor semen parameters and/or previous intracytoplasmic sperm injection (ICSI) failures with ejaculated spermatozoa. We compared the aneuploidy rate and quality between embryos derived from ICSI cycles with ejaculated sperm (EJ-ICSI) and those from ICSI cycles using testicular spermatozoa (TT-ICSI) within the same couple.Retrospective study of 27 couples who first underwent an EJ-ICSI cycle that did not result in a livebirth and afterwards a TT-ICSI cycle. Only the two closer cycles of each couple were included. Preimplantation genetic test for aneuploidies (PGT-A) was performed in both ICSI cycles and classic parameters of embryo quality were assessed until blastocyst-stage.A total of 375 embryos from 54 ICSI cycles were evaluated. Aneuploidy rate was measured by two different parameters. Patients undergoing TT-ICSI presented a similar aneuploidy rate as EJ-ICSI group: 30.7% (23.4-38.0) vs 26.8% (18.1-35.5) per inseminated oocytes (P0.05), and 76.2% (66.2-86.2) vs 72.1% (59.1-85.2) per the total number of biopsied embryos (P0.05), respectively. Further, the good-quality blastocyst rate per correctly fertilized oocyte was significantly higher in TT-ICSI group (33.6% (30.4-36.9)) than EJ-ICSI group (24.2% (20.3-28.0)) (P0.001).Switching to testicular sperm for ICSI yielded better-quality blastocysts without affecting the chromosomal load of the embryos in non-azoospermic couples with a previous unsuccessful ICSI using ejaculated sperm. This strategy is a good option for couples seeking a livebirth who do not want to use donor sperm.

Study question Is reproductive success measured as CLBR per inseminated oocyte, per embryo transfer and per embryo transferred affected by paternal age in donor egg IVF-ICSI cycles? Summary answer Paternal age does not significantly affect reproductive outcomes measured by CLBR per inseminated oocyte, embryo transfer and embryo transferred in donor egg IVF-ICSI cyles. What is known already In recent years, the delay in the start of formation of a family has led to an increase of the average male age at which the first child is conceived. Therefore, there is a growing interest on the study of the impact of male age on the reproductive outcomes in assisted reproduction cycles (ART). Several studies have evaluated the effect of paternal age on reproductive outcomes using donor egg cycles to control for female factors. However, the results obtained on this topic are still controversial leading to a need of more research about it, which this study tries to address. Study design, size, duration This retrospective observational multicentric cohort study has included donor IVF-ICSI treatments (n = 1539) performed to couples with etiology of male infertility (non-normozoospermic) in Spain IVIRMA clinics between January 2008 and March 2020 using patients’ own sperm sample. Paternal age ranged from 28 to 74 years. The study population was categorized in 5 groups following the criterion of homogenizing the number of observations between groups 28–38 (A), 38–41 (B), 41–44 (C), 44–48 (D) y 48–74 (E) years. Participants/materials, setting, methods Considering that male age could be a factor affecting reproductive outcomes, we evaluated men with different age that performed a donor IVF-ICSI treatment with their own semen, etiology of male infertility and known age. Data was exported in order to obtain the clinical database and Kaplan-Meier was used for data analysis. P Main results and the role of chance This study considered approximately 836 patients and 1411 embryo transfers. The CLBR per inseminated oocyte showed no significant difference between the study groups: A (3.09%, 42.61%, 71.96%), B (4.53%, 39.76%, 84.19%), C (5.89%, 47.04%, 78.61%), D (2.99%, 46.7%, 73.15%) and E (3.89%, 38.39%, 78.85%) for 7, 12 and 17 inseminated oocytes, respectively. In terms of CLBR per embryo transfer, the results obtained for each of the age groups were: A (51.55%, 70.69%, 92.18%), B (50.40%, 78.13%, 100.00%), C (53.68%, 71.69%, 100.00%), D (51.71%, 79.72%, 100.00%) and E (46.69%, 60.02%, 70.01%) for 3, 5 and 7 embryo transfers, respectively. No statistically significant differences were found among the studied age groups. CLBR per embryo transferred also did not show statistically significant differences between the age groups: A (8.43%, 53.34%, 72.13%), B (8.55%, 44,67%, 71.65%), C (10.40%, 53.94%, 72.49%), D (7.25%, 43.61%, 75.12%) and E (8.21%, 45.99%, 64.55%) for 1, 4 and 7 embryos transferred, respectively. Therefore, no significant differences were found in the number of inseminated oocytes, embryo transfers and embryos transferred needed to achieve a live birth between the age groups (p > 0.05), suggesting that maybe paternal age has no relevant clinical effect on donor egg cycles with our categorization. Limitations, reasons for caution The retrospective nature of this study leads to biases derived from the clinical practice and to the presence of missing data (limiting sample size). Moreover, this study included donor egg cycles for controlling female factors, so this limits the generalization of our results to a population of young women. Wider implications of the findings: Our study showed that the reproductive success measured as CLBR per embryo transfer, embryo transferred and inseminated oocytes was not statistically significant different among the studied age groups in donor egg cycles. Therefore, considering our study setting, paternal age does not affect reproductive success, however further studies should be done. Trial registration number NA

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

Study question Does the selection of non-apoptotic sperm via magnetic-activated cell sorting (MACS) reduce the aneuploidy rate of embryos from couples undergoing ICSI cycles with PGT-A using the patients’ own oocytes? Summary answer It does. The aneuploidy rate in the MACS group was 4.34% lower than the one obtained using semen samples processed according to standard clinical practice. What is known already MACS is a successful tool in eliminating proapoptotic sperm from a semen sample. However, the true effect of this technique on reproductive outcomes and the quality of the resulting embryos are a matter of controversy. Some studies report that its use improves the percentage of good quality blastocysts in women older than 30 years old compared to standard ICSI. Randomized clinical trials that compare MACS to a control sample consider parameters of embryo quality such as morphology at day 3 or day 5, symmetry of the blastomeres, blastocysts’ stage of expansion, but they do not consider embryo ploidy. Study design, size, duration Retrospective, multicentre, observational cohort study. 14,145 patients and 18,710 cycles were evaluated in the reference group. In the MACS group, 615 patients and 974 cycles were considered. Data were exported from cycles performed in Spanish IVIRMA clinics between January 2008 and February 2020. Participants/materials, setting, methods Unselected males in couples undergoing PGT-A cycles, then subdivided into male factor (MF) - total progressive motile sperm count lower than 5 million - and non-male factor (NMF) infertility. Statistical analysis performed using R v.4.0.0. Means were calculated and compared using two-tailed paired t-test, while proportions were compared using Fisher’s exact test and the chi-squared test and the appropriate correction for multiple comparisons. The aneuploidy rates for each group were compared using Fisher’s exact test. Main results and the role of chance In the control group 73,228 biopsied embryos, from which 71,439 were informative in the PGT-A. In the MACS group 3,919 biopsied embryos, from which 3,843 were informative. The aneuploidy rate, computed per informative embryo, was 68.87% (68.40%, 69.34%) in the reference group and 64.53% (62.43%, 66.64%) in the MACS group. Both comparisons were statistically significant (p-value ˂0.00001). According to these results, an embryo in the PGT-A programme using non-apoptotic sperm selected through MACS and autologous oocytes had a 5% less chance of being aneuploid than those embryos fertilised with standardly selected sperm (relative risk of 0.95 (0.91–0.98) p = 0.006769). Embryos conceived from NMF patients whose semen had been processed using MACS had a 4.27% lower aneuploidy rate than the reference (65.52% (63.16%, 67.88%) vs 69.79% (69.20%, 70.37%) respectively). This difference was statistically significant. Those embryos conceived using semen from patients with MF using MACS also showed a lower aneuploidy rate than the reference with MF (0.28% (55.48%, 65.08%) vs (64.94% (63.35%, 66.23%) respectively), although this difference was not statistically significant. Thus, the decrease in aneuploidy rate observed when comparing MACS and reference groups undergoing PGT-A cycles using autologous oocytes remained approximately the same in both MF and NMF semen samples. Limitations, reasons for caution The retrospective nature of the study subjects the data to biases or inaccuracies in their annotation in the clinics’ informatic platform from which they were exported. However, the statistical analysis aimed at controlling these biases as much as possible. Wider implications of the findings: The vast amount of data compiled for this study confirms that the selection of non-apoptotic sperm through MACS slightly decreases the aneuploidy rate of embryos compared to semen samples processed according to the clinics’ standards. This would be interesting for patients who are considering undergoing PGT-A cycles in the future. Trial registration number Not applicable

Simple Summary Many couples attending infertility clinics still need to repeat treatments and undergo several failed attempts before achieving a healthy newborn, which leaves room for improvement in the techniques we currently use in the clinic. Among the different procedures susceptible to improvement, the selection of the most adequate sperm to be injected inside the egg is crucial to the cycle’s success. Magnetic-activated cell sorting (MACS) is a technique that removes physiologically abnormal sperm that have started a programmed cell death (apoptotic) process from a semen sample. However, it is not recommended to all patients because there is no agreement between the published literature on whether it improves reproductive outcomes. This study used data from all intracytoplasmic sperm injection cycles performed using the patient’s own oocytes in our clinics from January 2008 to February 2020. Our findings support that MACS should not be recommended to all infertile couples, since there was no significant difference in results compared to treatments in which MACS was not used. This study provides clinicians and patients with more accurate information on how MACS will impact their chances of pregnancy, and it will lead to studies focused on specific populations to which the technique can be particularly helpful. Abstract The application of MACS non-apoptotic sperm selection in infertility clinics is controversial since the published literature does not agree on its effect on reproductive outcomes. Therefore, it is not part of the routine clinical practice. Classical measures of reproductive success (pregnancy or live birth rates per ovarian stimulation) introduce a bias in the evaluation of a technique’s effect, since only the best embryo is transferred. This retrospective, multicenter, observational study evaluated the impact of MACS on reproductive outcomes, measuring results in classical parameters and cumulative live birth rates (CLBR). Data from ICSI cycles using autologous oocyte in Spanish IVIRMA fertility clinics from January 2008 to February 2020 were divided into two groups according to their semen processing: standard practice (reference: 46,807 patients) versus an added MACS sperm selection (1779 patients). Only when measured as CLBR per embryo transferred and per MII oocyte used was the difference between groups statistically significant. There were no significant differences between MACS and reference groups on pregnancy and live birth rates. In conclusion, results suggest that non-apoptotic sperm selection by MACS on unselected males prior to ICSI with autologous oocytes has limited clinical impact, showing a subtle increase in CLBR per embryo transferred.

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.