Renin Angiotensin System, COVID-19 and Male Fertility: Any Risk for Conceiving?

The current knowledge concerning the connection between severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the renin-angiotensin system (RAS) system in the male reproductive apparatus is still limited, so dedicated studies are urgently required. Concerns about the male fertility consequences of SARS-CoV-2 infection have started to emerge, since epidemiologic studies observed that this coronavirus affects male patients more frequently and with increased severity, possibly because of the hormone-regulated expression of angiotensin-converting enzyme 2 (ACE2) receptor. A disturbance in fertility is also expected based on studies of the previous SARS-CoV infection, which targets the same ACE2 receptor when entering the host cells. In addition, bioinformatics analyses reveal the abundant expression of ACE2 receptor in the male reproductive tissues, particularly in the testis. It has been proposed that pharmacological intervention favoring the angiotensin-(1-7)/ACE2/Mas receptor pathway and increasing ACE2 expression and activity could greatly prevent inflammatory lesions in this area. Finally, in laboratories performing assisted reproductive technologies it is recommended that more attention should be paid not only to sperm quality but also to safety aspects. Data about the potential infectivity of seminal fluid are in fact conflicting and do not exclude risks for both personnel and patients. The potential infectivity of SARS-CoV-2 in reproductive male tissues should be strongly considered and further investigated for the proper management of in vitro fertilization procedures.

Synchrotron radiation soft X-ray microscopy and low energy X-ray fluorescence to reveal elemental changes in spermatozoa treated with photobiomodulation therapy.

Male infertility is a worldwide clinical issue that increases the number of couples resorting to assisted reproductive technologies (ARTs) to achieve pregnancy. Photobiomodulation therapy (PBMT) is a promising technique that can biostimulate cells and tissues and it is currently successfully employed to enhance the sperm motility in vitro. Nevertheless, its use has been so far restricted to the research field. In the present work, we exploited two PBMT protocols at an 800 nm wavelength on sperm derived from infertile individuals, detecting an increase in sperm motility 1 hour after irradiation. Moreover, in order to add new information about the molecular effect of PBMT, the content of some light elements was evaluated using high resolution X-ray fluorescence imaging. Interestingly, an increase in Na content was detected in the irradiated samples, possibly suggesting a role of this element in sperm motility; indeed, a low Na content was previously correlated with a poor sperm quality, low semen volume, and modest fertilization rate. Amplifying the knowledge of PBMT in the ART field will expedite the translational potentiality of the PBMT use in clinical settings.

Study of the mechanical properties of fresh and cryopreserved individual human oocytes.

In assisted reproduction technologies, the cryopreservation of oocytes is a common procedure used to circumvent female infertility. However, some morphological and functional alterations of oocytes have been observed depending on the protocol applied. In this work, the mechanical response of individual human oocytes before and after a freeze-thawing procedure was characterised. Oocytes, immediately after retrieval, were morphologically evaluated by bright-field optical microscopy and their elasticity measured by indentation measurements using atomic force microscopy. Oocytes were then frozen according to the open-vitrification protocol and stored in liquid nitrogen. Afterwards, the same oocytes were thawed and the indentation measurements repeated. Using this approach, we can follow the elasticity of a set of single oocytes from retrieval up to the freeze-thawing procedure. The analysis of the resulting data shows that the retrieved healthy oocytes, which preserve their healthy morphological features after cryopreservation, maintain unchanged also in stiffness values. In contrast, oocytes having dysmorphic characteristics, before and/or after freeze-thawing, show significant variations in their mechanical response. In addition, the dysmorphic oocytes are generally observed to be softer than the healthy oocytes. Our results indicate that stiffness of healthy oocytes is not considerably affected by the open-vitrification-thawing procedure, and that distinct elasticity ranges can be identified for healthy and dysmorphic oocytes. These findings indicate that the mechanical characterization of oocytes represents an opportunity to detect cellular defects, and assess the quality and bio-viability of processes such as cryopreservation.

Planar AFM macro-probes to study the biomechanical properties of large cells and 3D cell spheroids.

The ability to measure mechanical response of cells under applied load is essential for developing more accurate models of cell mechanics and mechanotransduction. Living cells have been mechanically investigated by several approaches. Among them, atomic force microscopy (AFM) is widely used thanks to its high versatility and sensitivity. In the case of large cells or 3D multicellular aggregates, standard AFM probes may not be appropriate to investigate the mechanical properties of the whole biological system. Owing to their size, standard AFM probes can compress only a single somatic cell or part of it. To fill this gap, we have designed and fabricated planar AFM macro-probes compatible with commercial AFM instruments. The probes are constituted of a large flat compression plate, connected to the chip by two flexible arms, whose mechanical characteristics are tuned for specific biological applications. As proof of concept, we have used the macro-probes to measure the viscoelasticity of large spherical biological systems, which have a diameter above 100 µm: human oocytes and 3D cell spheroids. Compression experiments are combined with visual inspection, using a side-view configuration imaging, which allows us to monitor the sample morphology during the compression and to correlate it with the viscoelastic parameters. Our measurements provide a quantitative estimate of the relaxation times of such biological systems, which are discussed in relation to data present in literature. The broad applicability of the AFM macro-probes can be relevant to study the biomechanical features in any biological process involving large soft materials. STATEMENT OF SIGNIFICANCE: The understanding of the role of physical factors in defining cell and tissue functions requires to develop new methods or improve the existing ones to accurately measure the biomechanical properties. AFM is a sensitive and versatile tool to measure the mechanical features from single proteins to single cells. When cells or cell aggregates exceed few tens of microns, AFM suffers from limitations. On these biological systems the control of the contact area and the application of a precise uniform compression becomes crucial. A modification of the standard cantilevers fabrication allowed us obtaining AFM macro-probes, having large planar contact area and spring constant suitable for biological investigations. They were demonstrated valuable to characterize the mechanical properties of large hierarchical biological systems.

Human ß-defensin 1 in follicular fluid and semen: impact on fertility.

PURPOSE: ß-defensins are antimicrobial peptides expressed at mucosal level of male and female genito-urinary tract, where they exert protective functions against infections, possibly preserving human health and fertility. In our study, we investigated the possible involvement of ß-defensins in female and male infertility in Italian infertile couples (i) evaluating the presence of human ß-defensin 1 (hBD-1) in follicular fluid (FF) and its correlation with in vitro fertilization (IVF) outcomes; (ii) investigating the relationship between hBD-1 levels in semen and IVF outcomes (comprising correlation with sperm parameters); and (iii) exploring the effect of hBD-1 peptide on spermatozoa motility in vitro. METHODS: A perspective observational analytic pilot study was conducted. hBD-1 concentration was measured with ELISA assay in FF and semen from 50 couples that underwent assisted procreation technique procedures due to infertility status. Moreover, hBD-1 exogenous peptide was administered to 29 normozoospermic semen and their motility was recorded. RESULTS: hBD-1 was detected in FF and its levels were significantly higher in women with good fertilization rate (≥ 75%), respect to those with a poor fertilization rate (< 75%). The hBD-1 semen concentrations in oligo-asthenozoospermic subjects were significantly lower than that in normozoospermic men. Instead, hBD-1 level in sperm and FF not correlated with pregnancy rate. Finally, incubation of sperm with exogenous hBD-1 significantly increased progressive motility after 1 h and 24 h. CONCLUSIONS: Being aware of the relatively small sample size and medium power, our results possibly suggest that hBD-1 could influence oocyte and sperm quality, and could improve, when exogenously added, sperm motility.

Light element distribution in fresh and frozen-thawed human ovarian tissues: a preliminary study.

RESEARCH QUESTION: Does synchrotron X-ray fluorescence (XRF) provide novel chemical information for the evaluation of human ovarian tissue cryopreservation protocols? DESIGN: Tissues from five patients undergoing laparoscopic surgery for benign gynaecological conditions were fixed for microscopic analysis either immediately or after cryopreservation. After fixation, fresh and slowly frozen samples were selected by light microscopy and transmission electron microscopy, and subsequently analysed with synchrotron XRF microscopy at different incident energies. RESULTS: The distributions of elements detected at 7.3 keV (S, P, K, Cl, Fe, and Os) and 1.5 keV (Na and Mg) were related to the changes revealed by light microscopy and transmission electron microscopy analyses. The light elements showed highly informative findings. The S distribution was found to be an indicator of extracellular component changes in the stromal tissues of the freeze-stored samples, further revealed by the transmission electron microscopy analyses. Low-quality follicles, frequent in the freeze-thawed tissues, showed a high Na level in the ooplasm. On the contrary, good-quality follicles were detected by a homogeneous Cl distribution. The occurrence of vacuolated follicles increased after cryopreservation, and the XRF analyses showed that the vacuolar structures contained mainly Cl and Na. CONCLUSIONS: The study demonstrates that elemental imaging techniques, particularly revealing the distribution of light elements, could be useful in establishing new cryopreservation protocols.

Investigating the mechanical properties of zona pellucida of whole human oocytes by atomic force spectroscopy.

The role of mechanics in numerous biological processes is nowadays recognized, while in others, such as the fertilization process, it is still neglected. In the case of oocytes the description of their mechanical properties could improve the comprehension of the oocyte-spermatozoon interaction and be helpful for application in in vitro fertilization (IVF) clinics. Herein the mechanical properties of whole human oocytes (HOs) immediately after retrieval are investigated by indentation measurements with atomic force spectroscopy under physiological conditions. Measurements are performed on immature (metaphase I - MI) and mature (metaphase II - MII) HOs. According to their morphological characteristics MII-HOs are classified as "suitable" and "rejected"; these latter would be usually rejected for intracytoplasmic sperm injection (ICSI). For all maturation stages we observe that the elastic response of the zona pellucida (ZP) outer layer was different and distinguishable from the rest of the ZP-HO. The elasticity of this ZP outer layer varies with maturation and quality: stiffness decreases from immature MI to good quality MII, up to poor-quality rejected MII. An indirect analysis with IVF outcome indicates that the ZP outer layer of analysed HOs donated by women who achieved pregnancy is stiffer than that of HOs from women with negative outcome. Our findings suggest that mechanical properties can represent important oocyte quality indicators that may be exploited for the design of innovative ICSI dedicated cell sorters.

Pitfalls and promises in FTIR spectromicroscopy analyses to monitor iron-mediated DNA damage in sperm.

Many drugs, chemicals, and environmental factors can impair sperm functionality by inducing DNA damage, one of the important causes of reduced fertility potential. The use of vibrational spectromicroscopy represents a promising approach for monitoring DNA integrity in sperm, although some limitations exist, depending from the experimental conditions. Here, we report that when using FTIR spectromicroscopy to reveal oxidative stress mediated by Fenton's reaction on hydrated sperm samples, DNA damage interpretation is partially compromised by unexpected cell surface precipitates. The precipitates give a broad band in the 1150-1000cm(-1) infrared region, which partially covers one of the signatures of DNA (phosphate stretching bands), and are detected as iron and oxygen containing material when using XRF spectroscopy. On the other hand, the analyses further support the potential of FTIR spectromicroscopy to reveal cellular oxidative damage events such as lipid peroxidation, protein misfolding and aggregations, as well as DNA strain breaks.

Comparative proteomic analysis of spermatozoa isolated by swim-up or density gradient centrifugation.

BACKGROUND: Reports about the morphologic and functional characteristics of spermatozoa prepared by density gradient centrifugation (DC) or swim-up (SU) have produced discordant results. We have performed a proteomic comparison of cells prepared by DC and SU providing a molecular insight into the differences between these two methods of sperm cell isolation. METHODS: Protein maps were obtained by 2-dimensional (2-D) separations consisting of isoelectrofocusing (IEF) from pI 3 to 11 followed by SDS-polyacrylamide gel electrophoresis. 2-D gels were stained with Sypro Ruby. Map images of DC and SU spermatozoa were compared using dedicated software. Intensities of a given spot were considered different between DC and SU when their group mean differed by >1.5-fold (p<0.05, Anova). RESULTS: No differences were observed for 853 spots, indicating a 98.7% similarity between DC and SU. Five spots were DC>SU and 1 was SU>DC. Proteins present in 3 of the differential spots could be identified. One DC>SU spot contained lactate dehydrogenase C and gamma-glutamylhydrolase, a second DC>SU spot contained fumarate hydratase and glyceraldehyde-3-phosphate dehydrogenase-2, and a SU>DC spot contained pyruvate kinase M1/M2. CONCLUSIONS: The differences in protein levels found on comparison of DC with SU spermatozoa indicate possible dissimilarities in their glycolytic metabolism and DNA methylation and suggest that DC cells may have a better capacitation potential.

Effect of seminal leukocytes on in vitro fertilization and intracytoplasmic sperm injection outcomes.

OBJECTIVE: To investigate the influence of seminal leukocytes on conventional IVF and intracytoplasmic sperm injection (ICSI) outcomes, using a flow cytometry method. DESIGN: Prospective study. SETTING: Tertiary infertility center and research institute. PATIENT(S): One hundred sixty-four couples undergoing conventional IVF or ICSI. INTERVENTION(S): Seminal leukocytes were counted by flow cytometry. MAIN OUTCOME MEASURE(S): Correlation between seminal leukocytes concentration and reproductive outcomes in IVF and ICSI cycles. RESULT(S): The median number of oocytes retrieved, the fertilization and cleavage rate, the median number and grade of embryos transferred, the median number of good-quality embryos transferred, and the median percentage of good-quality embryos from total embryos transferred, in leukocytospermic and non-leukocytospermic patients were not statistically different after either IVF or ICSI. Similarly, there were no significant differences between the two groups for implantation rate and clinical pregnancy rate. Multivariate logistic regression analysis showed that the reproductive outcomes were not influenced by adjustment for female age, infertility diagnosis, number of previous attempts, treatment protocol (GnRH agonist or antagonist), assisted reproduction procedure (IVF or ICSI), and leukocytospermia. By profiling the proper Poisson regression models, no leukocytospermia cut-off value was able to identify the subjects at risk for oocyte fertilization or embryo cleavage failure. CONCLUSION(S): Using a flow cytometry method, we demonstrated that leukocytospermia does not significantly influence IVF or ICSI outcomes. The same results were obtained by using lower or higher cut-off values for leukocytospermia (from 0.2 to 2 × 10(6)/mL).

Comparative analysis of the seminal plasma proteomes of oligoasthenozoospermic and normozoospermic men.

A comparative proteomic study of oligoasthenozoospermic and normozoospermic seminal plasmas was conducted to establish differences in protein expression. Oligoasthenozoospermia (when semen presents with a low concentration and reduced motility of spermatozoa) is common in male infertility. Two-dimensional protein maps from seminal plasma samples from 10 men with normozoospermia and 10 men with idiopathic oligoasthenozoospermia were obtained by isoelectric focusing followed by sodium dodecyl-sulphate polyacrylamide electrophoresis. Map images were analysed using dedicated software involving normalization, spot-to-spot volume comparison and statistical treatment of the results to establish the significance of differences between normal and oligoasthenozoospermic samples. Six out of 1028 spots showed over 1.5-fold relative intensity differences (P < 0.05, analysis of variance). Four proteins were identified by nano liquid chromatography-electrospray ionization-mass spectrometry/mass spectrometry of their tryptic peptides and database searches. Two proteins were more than three-fold under-expressed in oligoasthenozoospermia, namely epididymal secretory protein E1 and galectin-3-binding protein; the other (lipocalin-1 and a prolactin-inducible protein form) were over-expressed. The identity and differential expression of epididymal secretory protein E1 was verified by Western-blotting. The statistically significant differential expression of these four proteins in oligoasthenozoospermia compared with normozoospermia provides a molecular basis for further investigations into the pathogenic mechanisms underlying idiopathic oligoasthenozoospermia.

The application of scanning near field optical imaging to the study of human sperm morphology.

BACKGROUND: The morphology of spermatozoa is a fundamental aspect to consider in fertilization, sperm pathology, assisted reproduction and contraception. Head, neck, midpiece, principal and terminal part of flagellum are the main sperm components to investigate for identifying morphological features and related anomalies. Recently, scanning near-field optical microscopy (SNOM), which belongs to the wide family of nanoscopic techniques, has opened up new routes for the investigation of biological systems. SNOM is the only technique able to provide simultaneously highly resolved topography and optical images with a resolution beyond the diffraction limit, typical of conventional optical microscopy. This offers the advantage to obtain complementary information about cell surface and cytoplasmatic structures. RESULTS: In this work human spermatozoa both healthy and with morphological anomalies are analyzed by SNOM, to demonstrate the potentiality of such approach in the visualization of sperm morphological details. The combination of SNOM topography with optical (reflection and transmission) images enables to examine typical topographic features of spermatozoa together with underlying cytoplasmic structures. Indeed the head shape and inner components as acrosome and nucleus, and the organization of mitochondria in the midpiece region are observed. Analogously for principal tract of the tail, the ridges and the columns are detected in the SNOM topography, while their internal arrangement can be observed in the corresponding SNOM optical transmission images, without requiring specific staining procedures or invasive protocols. CONCLUSIONS: Such findings demonstrate that SNOM represents a versatile and powerful tool to describe topographical and inner structural details of spermatozoa simultaneously. This analysis could be helpful for better characterizing several morphological anomalies, often related to sperm infertility, which cannot be examined by conventional techniques all together.

Medical treatments for endometriosis-associated pelvic pain.

The main sequelae of endometriosis are represented by infertility and chronic pelvic pain. Chronic pelvic pain causes disability and distress with a very high economic impact. In the last decades, an impressive amount of pharmacological agents have been tested for the treatment of endometriosis-associated pelvic pain. However, only a few of these have been introduced into clinical practice. Following the results of the controlled studies available, to date, the first-line treatment for endometriosis associated pain is still represented by oral contraceptives used continuously. Progestins represent an acceptable alternative. In women with rectovaginal lesions or colorectal endometriosis, norethisterone acetate at low dosage should be preferred. GnRH analogues may be used as second-line treatment, but significant side effects should be taken into account. Nonsteroidal anti-inflammatory drugs are widely used, but there is inconclusive evidence for their efficacy in relieving endometriosis-associated pelvic pain. Other agents such as GnRH antagonist, aromatase inhibitors, immunomodulators, selective progesterone receptor modulators, and histone deacetylase inhibitors seem to be very promising, but there is not enough evidence to support their introduction into routine clinical practice. Some other agents, such as peroxisome proliferator activated receptors-γ ligands, antiangiogenic agents, and melatonin have been proven to be efficacious in animal studies, but they have not yet been tested in clinical studies.

Association between the JC polyomavirus infection and male infertility.

In recent years the incidence of male infertility has increased. Many risk factors have been taken into consideration, including viral infections. Investigations into viral agents and male infertility have mainly been focused on human papillomaviruses, while no reports have been published on polyomaviruses and male infertility. The aim of this study was to verify whether JC virus and BK virus are associated with male infertility. Matched semen and urine samples from 106 infertile males and 100 fertile males, as controls, were analyzed. Specific PCR analyses were carried out to detect and quantify large T (Tag) coding sequences of JCV and BKV. DNA sequencing, carried out in Tag JCV-positive samples, was addressed to viral protein 1 (VP1) coding sequences. The prevalence of JCV Tag sequences in semen and urine samples from infertile males was 34% (72/212), whereas the BKV prevalence was 0.94% (2/212). Specifically, JCV Tag sequences were detected in 24.5% (26/106) of semen and 43.4% (46/106) of urine samples from infertile men. In semen and urine samples from controls the prevalence was 11% and 28%, respectively. A statistically significant difference (p<0.05) in JCV prevalence was disclosed in semen and urine samples of cases vs. controls. A higher JC viral DNA load was detected in samples from infertile males than in controls. In samples from infertile males the JC virus type 2 strain, subtype 2b, was more prevalent than ubiquitous type 1. JCV type 2 strain infection has been found to be associated with male infertility. These data suggest that the JC virus should be taken into consideration as an infectious agent which is responsible for male infertility.

Factor V Leiden and prothrombin gene G20210A mutation and in vitro fertilization: prospective cohort study.

BACKGROUND: The influence of thrombophilia on fertility and on IVF outcome is very controversial. The objectives of this study were: (i) to compare the prevalence of Factor V Leiden (FVL) and prothrombin gene G20210A mutation (PGM) in women undergoing IVF to women with spontaneous pregnancy; (ii) to compare the IVF outcomes and the risk of complications in FVL and PGM carrier to non-carrier women. METHODS: From March 2005 to December 2009, a total of 510 women requiring IVF were recruited in a prospective cohort study. A separate population of 490 nulliparous women who conceived naturally was also evaluated as fertile controls. All women were tested for the presence of FVL and PGM. RESULTS: The prevalence of thrombophilic mutations was the same among women requiring IVF (6.9%) and women with spontaneous pregnancy (6.9%). A total of 480 patients underwent 1105 IVF cycles. There were 30 women carriers (86 IVF cycles) and 450 non-carriers for thrombophilic mutations (1019 IVF cycles). No significant differences in the mean number of oocytes retrieved and the number of good quality embryos transferred were found between the mutation carrier and non-mutation carrier women; likewise the reproductive outcome and the IVF complications were not statistically different between the two groups. The cumulative live birth rate after six IVF cycles was similar in the mutation carrier and non-mutation carrier women. For the mutation carrier women, the optimistic estimate of cumulative live birth rate after six IVF cycles was 60.8% and the conservative estimate was 50.0%. Corresponding rates for the non-mutation carrier women were 56.8 and 36.2%, respectively. CONCLUSIONS: The results of this study suggest that FVL and PGM presence in asymptomatic women and in the absence of other risk factors do not influence IVF outcome, or represent risk factors for ovarian hyperstimulation syndrome (OHSS), or favour thrombosis after IVF. Screening for FVL and PGM does not appear to be justified to identify the patients at the risk for IVF failure, and/or for OHSS, and/or for thrombotic complications.

Assisted reproduction treatment in severe myasthenia gravis.

OBJECTIVE: To report a case of a patient with a severe myasthenia gravis (MG) who underwent controlled ovarian hyperstimulation for assisted conception. DESIGN: Case report and literature review. SETTING: Tertiary infertility center. PATIENT(S): A 40-year-old woman affected by severe MG. INTERVENTION(S): Controlled ovarian hyperstimulation, oocyte retrieval, intracytoplasmic sperm injection (ICSI) procedure for severe oligoastenozoospermia. MAIN OUTCOME MEASURE(S): Short- and long-term effects of assisted reproduction treatment (ART) on the clinical course of MG. RESULT(S): A total of four ICSI cycles were performed. In the third cycle, a pregnancy was achieved, but a spontaneous abortion occurred. No changes in MG therapy were necessary, neither before nor after the treatment. All procedures were well tolerated and no exacerbations of symptoms occurred. By contrast a little, but persistent, improvement of clinical disease course was observed. CONCLUSION(S): This is the first report of a patient with severe MG who underwent ART cycles. Although more patients need to be evaluated, the present case suggests that MG patients should not be excluded a priori from ART.