The objective of this study was to evaluate the effect of the generation of reactive oxygen species (ROS) on the integrity of the DNA of human spermatozoa, and to determine if pretreatment with antioxidants can reduce DNA damage. Samples were obtained from 47 men undergoing infertility investigation. ROS were generated in the samples by the addition of xanthine/xanthine oxidase (X/XO) with or without antioxidants. After incubation at timed intervals (0-2 h) with X/XO, the percentage of spermatozoa with DNA fragmentation was determined using the method of TdT-mediated DNA end-labelling (TUNEL). Time intervals were selected to mimic the clinical situation in which spermatozoa are held for a period of time after swim-up while the oocytes are prepared for ICSI. A significant increase in sperm DNA damage was evident when samples were incubated in the presence of ROS for intervals of 1 and 2 h, but not when incubated with ROS for <1 h (P = 0.0001). The addition of antioxidants significantly decreased the amount of DNA damage induced by ROS generation (P < 0.04). ROS can cause an increase in DNA fragmentation and pretreatment with antioxidants can reduce DNA damage.

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The objective of the present study was to assess the integrity of maternal and/or paternal chromatin in injected oocytes that remained unfertilized after intracytoplasmic sperm injection (ICSI). The study was performed on 102 oocytes that failed to show pronuclear formation 18-20 h after ICSI. We used chromatin labelling with 4,6-diamidino-2-phenylindole (DAPI) to identify maternal and paternal chromatin, coupled with biotin-mediated end-labelling to assess DNA fragmentation in each gamete. It was shown that 50% of oocytes without pronuclear formation following ICSI contained chromatin with damaged DNA, and that the source of the DNA fragmentation was equally divided between the spermatozoon (25.8%) and the oocyte (24.4%). A significantly greater proportion of condensed spermatozoa in human oocytes had damaged DNA, compared to decondensed spermatozoa (24.7 compared to 5.9%, P=0.002). There was a significant increase in the incidence of DNA fragmentation in oocytes from patients older than 35 years (65+/-1.2%) compared to those from younger patients (36+/-1.0%) (P < 0.05). Further, 17% of unfertilized oocytes contained no paternal chromatin. Thus, DNA fragmentation in both spermatozoa and oocytes is associated with failure of fertilization in ICSI. In some cases of severe male factor infertility, a significant proportion of spermatozoa injected into oocytes may contain fragmented DNA. Injection of oocytes with spermatozoa containing abnormal chromatin will probably result in failure of sperm decondensation and fertilization. In older women, a significant proportion of oocytes injected may contain fragmented DNA. These observations may explain the consistent inability of most clinics to achieve fertilization rates higher than 65% with ICSI.

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Primate-parasite interactions are often investigated via coprological studies given ethical and conservation restrictions of collecting primate hosts. Yet, these studies are inadequate to recover adult helminths for taxonomic identification and to accurately assess their prevalence, intensity, abundance, and site of infection. Fresh carcasses found in anthropogenic landscapes come as informative and reliable alternatives. In this study, we identified the helminths of brown howler monkeys (Alouatta guariba clamitans) and their sites of infection, and measured their prevalence, intensity, and abundance of infection. We necropsied 18 adult males, 11 adult females, and 7 juvenile males that died in conflicts with the anthropogenic environment (domestic dog attacks, n = 11; electrocutions and road-kills, n = 10 each; unknown, n = 5) in periurban landscapes of southern Brazil between 2013 and 2019. We found three nematodes (Trypanoxyuris minutus, Dipetalonema gracile, and Parabronema bonnei) and one cestode (Bertiella cf. studeri), a diversity estimated to account for a sampling completeness of 99%. Prevalence ranged from 3% for P. bonnei to 100% for T. minutus. Mean abundance ranged from 2 (D. gracile and B. cf. studeri) to 55,116 (T. minutus) and mean intensity of infection ranged from 4 (B. cf. studeri) to 55,116 (T. minutus). Trypanoxyuris minutus sex ratio was strongly malebiased. The intensity of infection with T. minutus was higher in juvenile males and adult females than in adult males. The low parasite diversity and the helminths' mode of transmission are compatible with howlers' arboreality and folivorous-frugivorous diet. The howlers were not infected with soil-transmitted helminth parasites of humans and domestic animals on the ground and probably did not eat invertebrates to complement the diet. Given the lack of evidence of howler health problems, we suggest that the causes of death of the necropsied howlers are the major threats to the long-term conservation of the species at the study periurban landscapes.

Study question Can oocytes isolated from transgender men after oophorectomy support embryonic development? Summary answer Embryo developmental arrest at 4–8cell stage (day 3 embryos) indicates poor quality of in vitro matured oocytes from transgender men. What is known already Gender affirming surgery for transgender men leads to permanent infertility, as it involves bilateral oophorectomy. Current approaches for fertility preservation, such as oocyte freezing following ovarian hyperstimulation, may interfere with the wanted masculine characteristics and enhance gender dysphoria. In vitro matured oocytes (IVM) isolated following oophorectomy have been proposed as a source of potential gametes to ensure fertility preservation for transgender men with a child wish. From previous studies, it has been shown that these oocytes are able to undergo maturation and display normal spindles, but their competence to be fertilized and support embryonic development has not been addressed yet. Study design, size, duration We evaluated the quality of in vitro matured oocytes isolated from ovaries of transgender men by applying calcium imaging and monitoring fertilization and embryonic development following intracytoplasmic sperm injection (ICSI). Ovaries were collected in cold (4oC) or warm (37oC) medium, to investigate the best collection procedure. So far, results from four transgender men have been included. Participants/materials, setting, methods: Ovaries from four transgender men undergoing testosterone treatment were collected after oophorectomy in cold or warm medium. Cumulus oocyte complexes (COCs) were isolated and cultured in maturation medium for 48hrs. Mature oocytes were injected with donated sperm and assessed either by calcium imaging, measuring the total calcium release following injection, or following embryonic development. Donated in vitro matured oocytes, germinal vesicle(GV) or metaphase I(MI) origin, from other patients undergoing IVF treatment were used as controls. Main results and the role of chance In total, 179 COCs were collected from ovaries (n = 8) of four transgender men. From the COCs collected in warm medium, 73/105(69%) survived and 33/73(45%) reached metaphase II (MII). Of 21 MII injected with sperm, 13/21(62%) fertilized, 9/21(43%) formed 2 pronuclei (PN), 8/9(89%) reached the 2-cell stage, 3/9(33%) reached 4–8cell stage but arrested. From 74 COCs isolated in cold medium, 57/74(77%) survived and 28/57(49%) matured. Of the 11 MII injected with sperm, 7/11(64%) fertilized, 6/11(54%) formed 2PN, 6/6(100%) reached the 2-cell stage, 4/6(67%) reached 4–8cell but arrested. In the control group, 10/13 oocytes injected with the same sperm sample, were normally fertilized (77%), 8/10(80%) reached the 2-cell stage, 7/10(70%) reached the 4–8cell stage and 4/10(40%) became blastocysts. From the warm, cold and control conditions, respectively 12,14 and 17 MII oocytes were used for calcium imaging. The product of amplitude and frequency of calcium peaks, representing total calcium release, was calculated. Oocytes showed an average release of 0.66AU and 1.69AU for the warm and cold condition, respectively. The average value for control oocytes was 2.19AU. Limitations, reasons for caution One major limitation of our study is the lack of ovaries from cis women as control group. Our control oocytes originated from women undergoing IVF treatment and have undergone ovarian stimulation. Furthermore, the number of oocytes analysed and number of patients per group was limited and is being increased. Wider implications of the findings: Our data indicate that in vitro matured oocytes from transgender men ovaries display poor quality, as demonstrated by the poor embryonic development. In the future, we will apply nuclear transfer technology as a mean to overcome embryonic developmental arrest in this group of oocytes. Trial registration number Not applicable

Study question Do different ECMs/substrates and growth media culture conditions improve in vitro male human primordial germ cell (hPGC) expansion? Summary answer We achieved in vitro expansion improvement of male hPGCs with specific growth factors such as LIF, EGF, FGF2 and GDNF on gelatin- and vitronectin-coating cultures. What is known already PGCs are the precursors of male and female gametes, which are specified during early mammalian post-implantation embryonic development. PGCs undergo sequential cell divisions to differentiate into pro-spermatogonia (pSPG). In vitro propagation of pSPG could be important to understand the transition to spermatogonial stem cells (SSCs), important for fertility preservation in patients with infertility. Here, we aimed at performing a comparative analysis on in vitro feeder-free culture systems, based on different extracellular matrix (ECM) and growth media culture conditions, to support the expansion of the male germline stem cell populations using second trimester human male gonads as primary material. Study design, size, duration We collected human 2nd trimester male fetal gonads from elective abortions. Male gonads were dissected in saline solution (0.9% NaCl) and were either fixed overnight in 4% paraformaldehyde (PFA) for immunohistochemistry or disaggregated by enzymatic digestion for in vitro culture. Participants/materials, setting, methods After differential plating, fetal cells were cultured for 6 days. Disaggregated gonads were cultured with two different growing media (Medium 1 supplemented with LIF, EGF, FGF–2 and GDNF and Medium 2 supplemented with RA, BMP 4 and Activin A) on gelatin, laminin, vitronectin or matrigel coated plates. Cultured cells were immunostained, quantified for the expression of DDX4 and POU5F1 after 3 days (D3) and 6 days (D6) of culture. Main results and the role of chance We pursued to evaluate whether germ cells dissociated from a pool of male fetal gonads could propagate in vitro when cultured for D6 in different conditions. We observed that expansion of POU5F1-positive early PGCs and DDX4-positive late PGCs was only observed when cells were plated on gelatin or vitronectin and cultured with Medium 1, containing the growth factors LIF, EGF, FGF2 and GDNF. However, a reduced percentage of PGCs was observed in all four different coatings when grown with Medium 2, containing RA, BMP4 and Activin A. We analyzed the relative expression of the PGC markers POU5F1, DDX4 and MAGEA4 in histological sections of gonads from embryos at 18.5 weeks of gestation. Two populations of hPGCs were observed: ∼10–30% of the gonadal cells expressed solely DDX4 (late PGCs), whereas less than 10% of gonadal cells expressed POU5F1 (early PGCs). SOX9 and STARD1 expression was evaluated, confirming the presence of Sertoli cells and Leydig cells, respectively. Limitations, reasons for caution Due to the limited and difficulty to obtain human fetal tissue, a limited number of samples were used. Wider implications of the findings: We expanded human male fetal germ cells in vitro for D6 on gelatin and vitronectin coated plates with Medium 1, containing growth factors LIF, EGF, FGF2 and GDNF. Our findings provide a 2D culture system to expand hPGCs that could be useful to study propagation to pSPGs and eventually SSCs. Trial registration number Not applicable