Teresa Almeida-Santos

Not all sperm are equal: functional mitochondria characterize a subpopulation of human sperm with better fertilization potential.

Human sperm samples are very heterogeneous and include a low amount of truly functional gametes. Distinct strategies have been developed to characterize and isolate this specific subpopulation. In this study we have used fluorescence microscopy and fluorescence-activated cell sorting to determine if mitochondrial function, as assessed using mitochondrial-sensitive probes, could be employed as a criterion to obtain more functional sperm from a given ejaculate. We first determined that mitochondrial activity correlated with the quality of distinct human samples, from healthy donors to patients with decreased semen quality. Furthermore, using fluorescence-activated cell sorting to separate sperm with active and inactive mitochondria we found that this was also true within samples. Indeed, sperm with active mitochondria defined a more functional subpopulation, which contained more capacitated and acrosome intact cells, sperm with lower chromatin damage, and, crucially, sperm more able to decondense and participate in early development using both chemical induction and injection into mature bovine oocytes. Furthermore, cell sorting using mitochondrial activity produced a more functional sperm subpopulation than classic swim-up, both in terms of improvement in a variety of functional sperm parameters and in statistical significance. In conclusion, whatever the true biological role of sperm mitochondria in fertilization, mitochondrial activity is a clear hallmark of human sperm functionality.

Creating a Global Community of Practice for Oncofertility

Fertility preservation in the cancer setting, known as oncofertility, is a field that requires cross-disciplinary interaction between physicians, basic scientists, clinical researchers, ethicists, lawyers, educators, and religious leaders. Funded by the National Institutes of Health, the Oncofertility Consortium (OC) was formed to be a scientifically grounded, transparent, and altruistic resource, both intellectual and monetary, for building this new field of practice capable of addressing the unique needs of young patients with cancer. The OC has expanded its attention to include other nonmalignant conditions that can threaten fertility, and the work of the OC now extends around the globe, involving partners who together have created a community of shared effort, resources, and practices. The OC creates materials that are translated, disseminated, and amended by all participants in the field, and local programs of excellence have developed worldwide to accelerate the pace and improve the quality of oncofertility research and practice. Here we review the global oncofertility programs and the capacity building activities that strengthen these research and clinical programs, ultimately improving patient care.

Dual use of Diff-Quik-like stains for the simultaneous evaluation of human sperm morphology and chromatin status

BACKGROUND Sperm chromatin status and nuclear DNA damage can be detected using well-established assays. However, most techniques are time-consuming and/or involve elaborate protocols and equipment. We have recently developed a simple and fast method to monitor sperm chromatin status in field conditions using the Diff-Quik assay which is employed in fertility clinics to assess sperm morphology with standard bright field microscopy. In the present study, we demonstrate that any Diff-Quik-like stain can easily, reproducibly and routinely monitor human sperm chromatin status as well. METHODS Different Diff-Quik-like stains were used to assess sperm morphology and the presence of abnormal dark nuclear staining in human sperm from four ART centres. The TUNEL assay was performed in the same samples, and fertility outcomes were assessed. RESULTS A significant correlation was found between TUNEL-positive sperm and dark sperm nuclei. Moreover, associations were also found between the percentage of dark sperm nuclei and seminal parameters, embryo development rate, embryo quality and clinical pregnancy, as well as with cryptorchidism, and there was a tendency towards an association with age. A value of 32% abnormal staining is suggested as a predictive threshold for embryo development and pregnancy. CONCLUSIONS Our results show that any Diff-Quik-like stain, already implemented in most laboratories to assess sperm morphology, can be adapted as an indicator for chromatin status in human sperm.

Evaluation of human sperm chromatin status after selection using a modified Diff-Quik stain indicates embryo quality and pregnancy outcomes following in vitro fertilization

Sperm chromatin/DNA damage can be measured by a variety of assays. However, it has been reported that these tests may lose prognostic value in Assisted Reproductive Technology (ART) cycles when assessed in post‐prepared samples, possibly due to the normalizing effect promoted by sperm preparation procedures. We have recently implemented a modified version of the Diff‐Quik staining assay that allows for the evaluation of human sperm chromatin status in native samples, together with standard sperm morphology assessment. However, the value of this parameter in terms of predicting in vitro fertilization (IVF) and Intracytoplasmic sperm injection (ICSI) outcomes after sperm selection is unknown. In this study, data from 138 couples undergoing in vitro fertilization (IVF) or Intracytoplasmic sperm injection (ICSI) treatments showed that sperm chromatin integrity was significantly improved after density gradient centrifugation and swim up (p < 0.001), but no correlations were found with fertilization or embryo development rates (p > 0.05). However, sperm samples presenting lower percentages of damaged chromatin were associated with better quality (Grade I) embryos in both ART procedures (p < 0.05) and clinical pregnancy among IVF couples (p < 0.05). Furthermore, regression analysis confirmed the clinical value of Diff‐Quik staining in predicting IVF (but not ICSI) clinical pregnancy (OR: 0.927, 95% CI: 0.871–0.985, p = 0.015), and a threshold value of 34.25% for this parameter was established. The proportion of IVF couples achieving a clinical pregnancy was reduced 1.9‐fold when the percentage of abnormal dark staining was ≥34.25% (p = 0.05). In conclusion, the Diff‐Quik staining assay provides useful information regarding ART success, particularly in IVF cycles, where some degree of ‘natural’ sperm selection may occur; but not in ICSI, where sperm selection is operator dependent. This quick and low‐cost assay is suggested as an alternative method to detect sperm chromatin status in minimal clinical settings, when no other well‐established and robust assays (e.g. Sperm chromatin structure assay, terminal deoxynucleotidyl transferase‐mediated dUDP nick‐end labelling) are available.

Low amounts of mitochondrial reactive oxygen species define human sperm quality.

We have applied the mitochondria-specific superoxide fluorescent probe MitoSOX Red (MitoSOX) to detect mitochondria-specific reactive oxygen species (mROS) production in human sperm samples using flow cytometry. We show that human ejaculates are heterogeneous in terms of mROS production, with three subpopulations clearly detectable, comprising sperm that produce increasing amounts of mROS (MitoSOX-, MitoSOX+, and MitoSOX++). The sperm subpopulation producing the lowest amount of mROS represented the most functional subset of male gametes within the ejaculate, as it was correlated with the highest amount of live and non-apoptotic sperm and increased both in samples with better semen parameters and in samples processed by both density-gradient centrifugation and swim-up, both known to select for higher quality sperm. Importantly, the MitoSOX- subpopulation was clearly more prevalent in samples that gave rise to pregnancies following assisted reproduction. Our work, therefore, not only describe discreet human sperm heterogeneity at the mROS level but also suggests that mROS may represent a strategy to both evaluate sperm samples and isolate the most functional gametes for assisted reproduction.

Survey of Fertility Preservation Options Available to Patients With Cancer Around the Globe

Purpose Oncofertility focuses on providing fertility and endocrine-sparing options to patients who undergo life-preserving but gonadotoxic cancer treatment. The resources needed to meet patient demand often are fragmented along disciplinary lines. We quantify assets and gaps in oncofertility care on a global scale. Methods Survey-based questionnaires were provided to 191 members of the Oncofertility Consortium Global Partners Network, a National Institutes of Health–funded organization. Responses were analyzed to measure trends and regional subtleties about patient oncofertility experiences and to analyze barriers to care at sites that provide oncofertility services. Results Sixty-three responses were received (response rate, 25%), and 40 were analyzed from oncofertility centers in 28 countries. Thirty of 40 survey results (75%) showed that formal referral processes and psychological care are provided to patients at the majority of sites. Fourteen of 23 respondents (61%) stated that some fertility preservation services are not offered because of cultural and legal barriers. The growth of oncofertility and its capacity to improve the lives of cancer survivors around the globe relies on concentrated efforts to increase awareness, promote collaboration, share best practices, and advocate for research funding. Conclusion This survey reveals global and regional successes and challenges and provides insight into what is needed to advance the field and make the discussion of fertility preservation and endocrine health a standard component of the cancer treatment plan. As the field of oncofertility continues to develop around the globe, regular assessment of both international and regional barriers to quality care must continue to guide process improvements.Purpose Oncofertility focuses on providing fertility and endocrine-sparing options to patients who undergo life-preserving but gonadotoxic cancer treatment. The resources needed to meet patient demand often are fragmented along disciplinary lines. We quantify assets and gaps in oncofertility care on a global scale. Methods Survey-based questionnaires were provided to 191 members of the Oncofertility Consortium Global Partners Network, a National Institutes of Health–funded organization. Responses were analyzed to measure trends and regional subtleties about patient oncofertility experiences and to analyze barriers to care at sites that provide oncofertility services. Results Sixty-three responses were received (response rate, 25%), and 40 were analyzed from oncofertility centers in 28 countries. Thirty of 40 survey results (75%) showed that formal referral processes and psychological care are provided to patients at the majority of sites. Fourteen of 23 respondents (61%) stated that some fertility preservation services are not offered because of cultural and legal barriers. The growth of oncofertility and its capacity to improve the lives of cancer survivors around the globe relies on concentrated efforts to increase awareness, promote collaboration, share best practices, and advocate for research funding. Conclusion This survey reveals global and regional successes and challenges and provides insight into what is needed to advance the field and make the discussion of fertility preservation and endocrine health a standard component of the cancer treatment plan. As the field of oncofertility continues to develop around the globe, regular assessment of both international and regional barriers to quality care must continue to guide process improvements.

Survey of Third-Party Parenting Options Associated With Fertility Preservation Available to Patients With Cancer Around the Globe

Purpose In the accompanying article, “Survey of Fertility Preservation Options Available to Patients With Cancer Around the Globe,” we showed that specific fertility preservation services may not be offered at various sites around the world because of cultural and legal barriers. We assessed global and regional experiences as well as the legal status of third-party reproduction and adoption to serve as a comprehensive international data set and resource for groups that wish to begin oncofertility interventions. Methods We provide data on the legalities of third-party assisted reproductive technologies and other family-building options in the 28 oncofertility-practicing countries surveyed. Results We found regional and country differences that will be important in the development of tailored resources for physicians and for patient brochures that are sensitive to these local restrictions and cultural norms. Conclusion Because many patients first consult Web-based materials, the formal assessment of the availability of these options provides members of the global oncofertility community with data to which they might otherwise not have ready access to better serve their patients.Purpose In the accompanying article, “Analysis of Fertility Preservation Options Available to Patients With Cancer Around the Globe,” we showed that specific fertility preservation services may not be offered at various sites around the world because of cultural and legal barriers. We assessed global and regional experiences as well as the legal status of third-party reproduction and adoption to serve as a comprehensive international data set and resource for groups that wish to begin oncofertility interventions. Methods We provide data on the legalities of third-party assisted reproductive technologies and other family-building options in the 28 oncofertility-practicing countries surveyed. Results We found regional and country differences that will be important in the development of tailored resources for physicians and for patient brochures that are sensitive to these local restrictions and cultural norms. Conclusion Because many patients first consult Web-based materials, the formal assessment of the availability of these options provides members of the global oncofertility community with data to which they might otherwise not have ready access to better serve their patients.

Anterior positioning of sex chromosomes on the head of human sperm sorted using visible wavelengths.

The human ejaculate contains subpopulations of sperm with distinct properties. Human X- and Y-bearing sperm were separated with fluorescence activated cell sorting. To avoid the use of UV light the quantitative DNA dyes DRAQ5® and Dyecycle™ Vybrant® Violet were used. Sorting efficiency was similar for both dyes, but lower than what is usually obtained with the classical method involving Hoechst 33342 and UV light (60-70% enrichment, versus 80-90%). A total of 2,739 spermatozoa were evaluated, from seven distinct samples using fluorescence in situ hybridization (FISH) chromosomal probes. No differences were found in sorted and unsorted populations in terms of chromosome positioning, and numeric chromosomal anomalies were not more evident following cell sorting. Furthermore in both sorted and unsorted populations the sex chromosomes were clearly located in the anterior portion of the sperm head, while a control autosome (chromosome 18) showed no such tendency, confirming previous findings. These results suggest that other quantitative DNA dyes may be used for sex chromosome-based human sperm sorting, but with lower efficiency than the standard UV-Hoechst based assay.

Optimizing the Decision-Making Process About Fertility Preservation in Young Female Cancer Patients: The Experience of the Portuguese Centre for Fertility Preservation

The reproductive future of young and childless cancer patients is at risk, and fertility preservation (FP) methods are available to attempt to ensure their potential for biological parenthood after cancer treatments. Oncofertility is a new research and intervention field that unites health professionals from several fields to explore options for the reproductive future of cancer patients.

Are women and men well informed about fertility? Childbearing intentions, fertility knowledge and information-gathering sources in Portugal

BackgroundThe postponement of parenthood may increase the number of couples experiencing infertility and prolonged time to pregnancy. Previous research has revealed that childless people are not well informed regarding fertility, which may threat their childbearing intentions. This study aimed to examine fertility knowledge and childbearing intentions held by Portuguese people and their use and perceived usefulness of information sources on fertility.MethodsParticipants were recruited using a random-route domiciliary approach. A total of 2404 individuals aged 18–45 were asked to complete a structured questionnaire evaluating socio-demographic characteristics, childbearing intentions, fertility knowledge and information-gathering sources regarding fertility.ResultsIn total, 95.5% of the participants indicated the desire to have children in the future, and 61.7% reported that having children would contribute to life satisfaction. Most of the participants expressed the desire to have two children in the future. The discrepancy between the numbers of planned and desired children was higher in men, in participants with lower education levels, in professionally active participants and in the unemployed participants. Relationship stability seemed to be more important in influencing childbearing decisions than financial stability or family support.Participants’ knowledge regarding fertility was poor. Women, the participants who were older than 25, the participants with longer education and the participants with higher income exhibited the greatest levels of knowledge of fertility, although this knowledge was only slightly enhanced in these subgroups. Also, the participants overestimated both the chances of spontaneous pregnancy and the success rates of assisted reproduction techniques. Finally, the results revealed that websites were the main information sources used by the participants and only 18.0% of the participants had previously discussed fertility issues with their doctors.ConclusionsAlthough Portuguese men and women reported the desire to have children in the future, their knowledge regarding fertility and infertility risk was poor. In addition, participants used more general sources of information, such as website, but not specialized sources, such as their doctors. There is a real need to work with general practitioners to empower them to provide adequate fertility information to every childless patient.