Phase angle at bioelectric impedance analysis is associated with detrimental sperm quality in idiopathic male infertility: a preliminary clinical study.

Background: In 2020, 38% of adults were affected by obesity, while infertility globally affected 1 in 6 people at some stage of their lives.Body mass index (BMI) provides an easy but occasionally inaccurate estimation of body composition. To achieve a more precise assessment, bioelectric impedance analysis serves as a validated tool that administers electrical energy through surface electrodes. Phase angle as a function of the relationship between tissues resistance and reactance, is a trustworthy predictor of body composition and cell membrane integrity. Objectives: We aim to assess whether there is an association between phase angle and seminal parameters, as well as sperm DNA fragmentation percentage. Design: Semen samples of 520 idiopathic infertile patients were analyzed according to 2021 World Health Organization guidelines and evaluated for sperm DNA fragmentation rate. Each participants underwent bioelectric impedance analysis. Results: Median age was 40 years old, median BMI was 26.3 kg/m2, median phase angle was 6.2°. In the logistic regression analysis adjusted for age and total intracorporeal water, phase angle (continuous) was significantly associated with oligozoospermia (odds ratio [OR]:0.4; p<0.01) and sperm morphology (OR: 0.65; p=0.05) and slightly with sperm DNA fragmentation (OR: 0.98; p=0.07). In subgroup analysis, the logistic regression analysis adjusted for the mentioned parameters showed that a phase angle between 6.2 and 7 (°) (OR: 0.63; p=0.02) and >7 (°) (OR: 0.12; p<0.01) were associated with a reduced risk of oligozoospermia compared to values <6.2 (°). Similarly, a phase angle between 6.2 and 7 (°) (OR: 0.57; p< 0.01 and OR: 0.58; p= 0.01) and PA > 7 (°) (OR: 0.12; p= 0.03 and OR: 0.21; p< 0.01) were associated with a reduced risk of lower sperm concentration and lower total sperm count, respectively, compared to a phase angle < 6.2 (°). Conclusion: Our study suggests a negative association between phase angle and detrimental sperm parameters in male idiopathic infertility.

Diagnosis and management of infertility: NICE-adapted guidelines from the Italian Society of Human Reproduction.

In Italy the fertility rate is very low, and an increasing number of patients are infertile and require treatments. The Italian Law concerning the safety of patient care, and the professional liability of health professionals, indicates that health professionals must comply with the recommendations set out in the guidelines developed by public and private bodies and institutions, as well as scientific societies and technical-scientific associations of the health professions, except for specific cases. Unfortunately, no guideline for the diagnosis and the management of infertility is currently available in Italy. In 2019, the Italian Society of Human Reproduction pointed out the need to produce Italian guidelines and subsequently approved the establishment of a multidisciplinary and multiprofessional working group (MMWG) to develop such a guideline. The MMWG was representative of 5 scientific societies, one national federation of professional orders, 3 citizens' and patients' associations, 5 professions (including lawyer, biologist, doctor, midwife, and psychologist), and 3 medical specialties (including medical genetics, obstetrics and gynecology, and urology). The MMWG chose to adapt a high-quality guideline to the Italian context instead of developing one from scratch. Using the Italian version of the Appraisal of Guidelines for Research and Evaluation II scoring system, the National Institute of Clinical Excellence guidelines were selected and adapted to the Italian context. The document was improved upon by incorporating comments and suggestions where needed. This study presents the process of adaptation and discusses the pros and cons of the often-neglected choice of adapting rather than developing new guidelines.

What Does Intracytoplasmic Sperm Injection Change in Embryonic Development? The Spermatozoon Contribution.

The intracytoplasmic sperm injection (ICSI) technique was invented to solve severe male infertility due to altered sperm parameters. Nowadays, it is applied worldwide for the treatment of couple infertility. ICSI is performed with any available spermatozoon from surgery or ejaculated samples, whatever are the sperm motility, morphology or quantity. The aim of the present review was to study if embryo development and kinetics would be modified by (1) ICSI under the technical aspects, (2) the micro-injected spermatozoa in connection with male infertility. From published data, it can be seen that ICSI anticipates the zygote kinetics Furthermore, because fertilization rate is higher in ICSI compared to conventional in vitro fertilization (IVF), more blastocysts are obtained for clinical use in ICSI. Sperm and spermatozoa characteristics, such as sperm parameters, morphology and vitality, DNA content (levels of sperm DNA fragmentation, microdeletions, and chromosomal abnormalities), RNA content, epigenetics, and sperm recovery site (testicular, epididymis, and ejaculated), have an impact on fertilization and blastocyst rates and embryo kinetics in different ways. Even though ICSI is the most common solution to solve couples' infertility, the causes of male infertility are crucial in building a competent spermatozoa that will contribute to normal embryonic development and healthy offspring.

The paradox of the Italian clinical embryologist in the national public health system: hints towards harmonization of a postgraduate educational curriculum.

Clinical embryologists are highly trained laboratory professionals with multiple roles, including laboratory, clinical, biobanking and quality system management. In most European countries, clinical embryologists are trained to work in Medically Assisted Reproduction (MAR) centres without a specifically dedicated educational path. The criteria required for employment vary according to the educational structure and the public or private nature of the centre. We have herein described the educational profile required by Italian clinical embryologists to work in MAR centres of the National Health System (NHS). Public centres currently represent 36% of all the Italian MAR clinics. According to the Italian law, a future clinical embryologist must achieve a 3-4 year unpaid post-graduate specialization in a different field, choosing from Genetics, Microbiology, Clinical Pathology or Nutrition. Accesses to the above-mentioned post-graduate courses are themselves very limited. Clinical embryologists are basically trained by senior colleagues. This situation makes inevitably difficult to recruit laboratory staff in NHS centres. Moreover, it represents an emblematic example of the need for an equal training curriculum, possibly ensuring a comparable education quality, mobility of trainees and dissemination of skills for clinical embryologists all over Europe.

Couples undergoing Assisted Reproductive Techniques: An Actor-Partner Interdependence Model of dyadic adjustment, attachment, and body-image avoidance.

The aim is to assess the commonalities and interdependence referred to body-image avoidance among 118 couples newly introduced to first-level assisted reproductive techniques. Results showed non-clinical functioning levels, and partners showed a correlated, yet low, dyadic adjustment. The couple-effect was modeled through an Actor Partner Interdependence Model. For both partners, dyadic adjustment's actor-effect associates with body-image avoidance. Moreover, psychological symptoms' actor-effect associate to body-image avoidance, resulting significantly more influential than the partner-effect. Only for males, alexithymia's actor-effect was significant. To conclude, partners' functioning is quite specular yet not interdependent, as they do not show a couple-as-a-unit modality of functioning. Clinical implications are discussed.

Air Pollution and COVID-19: A Possible Dangerous Synergy for Male Fertility.

Several studies indicate that semen quality has strongly declined in the last decades worldwide. Air pollution represents a significant co-factor with the COVID-19 impact and has negative effects on the male reproductive system, through pro-oxidant, inflammatory and immune-dysregulating mechanisms. It has recently been reported that chronic exposure to PM2.5 causes overexpression of the alveolar ACE2 receptor, the entry route of SARS-CoV-2 into the organism shared by the lungs and testis where expression is highest in the body. In the testis, the ACE2/Ang-(1-7)/MasR pathway plays an important role in the regulation of spermatogenesis and an indirect mechanism of testicular damage could be due to the blockade of the ACE2 receptor by SARS-CoV-2. This prevents the conversion of specific angiotensins, and their excess causes inflammation with the overproduction of cytokines. PM2.5-induced overexpression of the alveolar ACE2 receptor, in turn, could increase local viral load in patients exposed to pollutants, producing ACE2 receptor depletion and compromising host defenses. By presenting an overall view of epidemiological data and molecular mechanisms, this manuscript aims to interpret the possible synergistic effects of both air pollution and COVID-19 on male reproductive function, warning that the spread of SARS-CoV-2 in the fertile years may represent a significant threat to global reproductive health. All of this should be of great concern, especially for men of the age of maximum reproductive capacity, and an important topic of debate for policy makers. Altered environmental conditions, together with the direct and indirect short- and long-term effects of viral infection could cause a worsening of semen quality with important consequences for male fertility, especially in those areas with higher environmental impact.

More blastocysts are produced from fewer oocytes in ICSI compared to IVF - results from a sibling oocytes study and definition of a new key performance indicator.

BACKGROUND: Which fertilization method, between ICSI and IVF in split insemination treatments, has the highest clinical efficiency in producing clinically usable blastocyst? METHODS: 211 infertile couples underwent split insemination treatments for a non-severe male factor. 1300 metaphase II (MII) oocytes were inseminated by conventional IVF and 1302 MII oocytes were micro-injected with the same partner's semen. Embryo development until blastocyst stage on day V and clinical outcomes were valuated trough conventional key performance indicators (KPI), and new KPIs such as blastocyst rate per used MII oocytes and the number of MII oocytes to produce one clinically usable blastocyst from ICSI and IVF procedures. RESULTS: The results were  globally analyzed and according to ovarian stimulation protocol, infertility indication, and female age. The conventional KPI were online with the expected values from consensus references. From global results, 2.3 MII oocyte was needed to produce one clinically usable blastocyst after ICSI compared to 2.9 MII oocytes in IVF. On the same way, more blastocysts for clinical use were produced from fewer MII oocytes in ICSI compared to IVF in all sub-groups. CONCLUSIONS: In split insemination treatments, the yield of clinically usable blastocysts was always superior in ICSI compared to IVF. The new KPI "number of needed oocytes to produce one clinically usable embryo" tests the clinical efficiency of the IVF laboratory.

Semen quality as a potential susceptibility indicator to SARS-CoV-2 insults in polluted areas.

The epidemic of the new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has impacted worldwide with its infectious spread and mortality rate. Thousands of articles have been published to tackle this crisis and many of these have indicated that high air pollution levels may be a contributing factor to high outbreak rates of COVID-19. Atmospheric pollutants, indeed, producing oxidative stress, inflammation, immuno-unbalance, and systemic coagulation, may be a possible significant co-factor of further damage, rendering the body prone to infections by a variety of pathogens, including viruses. Spermatozoa are extremely responsive to prooxidative effects produced by environmental pollutants and may serve as a powerful alert that signals the extent that environmental pressure, in a specific area, is doing damage to humans. In order to improve our current knowledge on this topic, this review article summarizes the relevant current observations emphasizing the weight that environmental pollution has on the sensitivity of a given population to several diseases and how semen quality, may be a potential indicator of sensitivity for virus insults (including SARS-CoV-2) in high polluted areas, and help to predict the risk for harmful effects of the SARS-CoV-2 epidemic. In addition, this review focused on the potential routes of virus transmission that may represent a population health risk and also identified the areas of critical importance that require urgent research to assess and manage the COVID-19 outbreak.

The true panel of cystic fibrosis mutations in the Sicilian population.

BACKGROUND: The aim was to establish the true risk of having an affected child with Cystic Fibrosis (CF) in the Sicilian infertile population. METHODS: A longitudinal CFTR screening of 1279 Sicilian infertile patients for all CFTR mutations sequencing the entire gene by Next Generation Sequencing (NGS) was performed from patient's blood. RESULTS: One patient out of 16 was a carrier of a CFTR mutation. Twenty-four mutations were found. Theoretically one couple out of 256 was at risk of CF transmission. CONCLUSIONS: The risk of CF transmission is unexpectedly high in Sicily and with a high heterogeneity. Sequencing an entire and long gene such as CFTR makes accessible the true panel of mutations in a specific population and helps better to understand the true risk of having an affected child.

Universal strategy for preimplantation genetic testing for cystic fibrosis based on next generation sequencing.

PURPOSE: We developed and applied a universal strategy for preimplantation genetic testing for all cystic fibrosis gene mutations (PGT-CF) based on next-generation sequencing (NGS). METHODS: A molecular protocol was designed to diagnose all CF mutations at preimplantation stage. The detection of CF mutations was performed by direct gene sequencing and linkage strategy testing 38 specific SNPs located upstream and inside the gene for PGT-CF. Seventeen couples at risk of CF transmission decided to undergo PGT-CF. Trophectoderm cell biopsies were performed on day 5-6 blastocysts. PGT for aneuploidy (PGT-A) was performed from the same samples. Tested embryos were transferred on further natural cycles. RESULTS: PGT was performed on 109 embryos. Fifteen CF mutations were tested. PGT-CF and PGT-A were conclusive for respectively 92.7% and 95.3% of the samples. A mean of 24.1 SNPs was informative per couple. After a single embryo transfer on natural cycle, 81.3% of the transferred tested embryos were implanted. CONCLUSIONS: The present protocol based on the entire CFTR gene together with informative SNPs outside and inside the gene can be applied to diagnose all CF mutations at preimplantation stage.

Universal strategy for preimplantation genetic testing for cystic fibrosis based on next-generation sequencing.

PURPOSE: We developed and applied a universal strategy for preimplantation genetic testing for all cystic fibrosis gene mutations (PGT-CF) based on next-generation sequencing (NGS). METHODS: A molecular protocol was designed to diagnose all CF mutations at preimplantation stage. The detection of CF mutations was performed by direct gene sequencing and linkage strategy testing 38 specific SNPs located upstream and inside the gene for PGT-CF. Seventeen couples at risk of CF transmission decided to undergo PGT-CF. Trophectoderm cell biopsies were performed on days 5-6 blastocysts. PGT for aneuploidy (PGT-A) was performed from the same samples. Tested embryos were transferred on further natural cycles. RESULTS: PGT was performed on 109 embryos. Fifteen CF mutations were tested. PGT-CF and PGT-A were conclusive for, respectively, 92.7% and 95.3% of the samples. A mean of 24.1 SNPs was informative per couple. After single embryo transfer on natural cycle, 81.3% of the transferred tested embryos implanted. CONCLUSIONS: The present protocol based on the entire CFTR gene sequencing together with informative SNPs outside and inside the gene can be applied to diagnose all CF mutations at preimplantation stage.

Sperm DNA fragmentation: An early and reliable marker of air pollution.

Environmental factors could have a key role in the continuous and remarkable decline of sperm quality observed in the last decades. This study compared the seminal parameters and sperm DFI in men living in areas with different levels of air pollution. Results demonstrate that both steel plants workers and patients living in a high polluted area show a mean percentage of sperm DNA fragmentation above 30%, highlighting a clear sperm damage. In this work, two different techniques were used to measure sperm DNA damage in patients' groups, finding in both cases a high sperm DFI in patients living in polluted areas. We candidate sperm DNA fragmentation as a valuable early marker of the presence and harmful effects of pollution. We suggest that sperm DNA evaluation could be both an indicator of individual health and reproductive capacity, and a suitable datum to connect the surrounding environment with its effects.

The decision on the embryo to transfer after Preimplantation Genetic Diagnosis for X-autosome reciprocal translocation in male carrier.

BACKGROUND: The aim of Preimplantation Genetic Diagnosis (PGD) on embryos produced in vitro is to identify the embryos without genetic or chromosomal defect from those embryos that will develop the genetic disease or are chromosomally abnormal. In case of PGD for structural chromosome indication (PGR-SR), the normal/balanced embryos are transferred in the maternal uterus. This protocol is valid and widely applied for autosomal chromosome translocation. But which embryo should be transferred after preimplantation genetic diagnosis (PGD-SR) for X-3 reciprocal translocation in male patient? CASE PRESENTATION: The female patient was 26 years old with normal 46,XX karyotype. The male patient had a karyotype with balanced translocation 46,Y,t(X;3)(p11.2;p14)mat, inherited from the mother. The female patient underwent two cycles of ovarian stimulation. In the first cycle, the metaphase II oocytes were vitrified, while in the second cycle they were used as fresh. ICSI was performed on vitrified/warmed and fresh oocytes. Embryos were biopsied at blastocyst stage. Chromosomal analysis was performed by Next Generation Sequencing.Eleven blastocysts were biopsied from 23 vitrified/warmed and fresh metaphase II oocytes. Two embryos were diagnosed 46,XY; two embryos were diagnosed 46,XX; four embryos were diagnosed with unbalanced translocations and three embryos were diagnosed aneuploid. We knew that the two embryos diagnosed as 46,XX inherited the balanced translocation from the father and the two embryos diagnosed as 46,XY had a normal karyotype. It was explain to the couple that the phenotype of balanced translocated female embryos cannot be predicted because of the random inactivation of X chromosome and that could also occur on the der(X). The couple asked to have a 46,XY embryo transferred. Clinical pregnancy was obtained and non invasive prenatal test confirmed PGD-SR result. CONCLUSIONS: Proposing PGD-SR for gonosome-autosome reciprocal translocation implies the risk to exclude balanced translocated female embryos with a normal phenotype for transfer because the early and late normal development at post-natal stage cannot be predicted based on the only chromosomal analysis.

Effects of Highly Polluted Environment on Sperm Telomere Length: A Pilot Study.

High environmental pressure may impair male fertility by affecting sperm quality, but the real effect remains controversial. Herein, we assessed the influence of environmental exposure on telomere length (TL) in both leukocytes (LTL) and sperm cells (STL). A pilot biomonitoring study was conducted in 112 clinically healthy, normospermic men living in various areas of Campania region (South of Italy) with high (n = 57, High Group) or low (n = 55, Low Group) environmental pressure. TL analysis was assessed by quantitative real time-PCR. STL was not significantly correlated with either age (p = 0.6) or LTL (p = 0.7), but was significantly longer in the High Group compared with the Low Group (p = 0.04). No significant difference was observed between leukocyte TL in the High or Low Group. Our results showed that male residents in areas with high environment exposure had a significant increase in STL. This finding supports the view that the human semen is a sentinel biomarker of environmental exposure.

The accumulation of vitrified oocytes is a strategy to increase the number of euploid available blastocysts for transfer after preimplantation genetic testing.

PURPOSE: In a preimplantation genetic diagnosis for aneuploidy (PGD-A) program, the more embryos available for biopsy, consequently increases the chances of obtaining euploid embryos to transfer. The aim was to increase the number of viable euploid blastocysts in patients undergoing PGD-A using fresh oocytes together with previously accumulated vitrified oocytes. METHODS: Sixty-nine patients with normal ovarian reserve underwent PGD-A for repeated implantation failure or recurrent pregnancy loss indication. After several cycles of ovarian stimulation, 591 accumulated vitrified oocytes and 463 fresh oocytes were micro-injected with the same partner's semen sample. PGD-A was completed on 134 blastocysts from vitrified/warmed oocytes and 130 blastocysts from fresh oocytes. RESULTS: A mean of 9.6% euploid blastocyst per micro-injected vitrified/warmed oocytes and 11.4% euploid blastocyst per micro-injected fresh oocyte were obtained (p > 0.05). The euploidy and aneuploidy rates were comparable in blastocysts obtained from micro-injected vitrified/warmed oocytes and fresh oocytes (42.5 versus 40.8% and 57.5 versus 59.2%, p > 0.05). Implantation rates of euploid blastocysts were comparable between the two sources of oocytes (56.0% from vitrified/warmed oocytes versus 60.9% from fresh oocytes, p > 0.05). CONCLUSIONS: Oocyte vitrification and warming do not generate aneuploidy in blastocysts. The number of viable euploid embryos for transfer can be increased by using accumulated vitrified oocytes together with fresh oocytes in ICSI. TRIAL REGISTRATION: NCT02820415 ClinicalTrials.gov.

Different Tissue-Derived Stem Cells: A Comparison of Neural Differentiation Capability.

BACKGROUND: Stem cells are capable of self-renewal and differentiation into a wide range of cell types with multiple clinical and therapeutic applications. Stem cells are providing hope for many diseases that currently lack effective therapeutic methods, including strokes, Huntington's disease, Alzheimer's and Parkinson's disease. However, the paucity of suitable cell types for cell replacement therapy in patients suffering from neurological disorders has hampered the development of this promising therapeutic approach. AIM: The innovative aspect of this study has been to evaluate the neural differentiation capability of different tissue-derived stem cells coming from different tissue sources such as bone marrow, umbilical cord blood, human endometrium and amniotic fluid, cultured under the same supplemented media neuro-transcription factor conditions, testing the expression of neural markers such as GFAP, Nestin and Neurofilaments using the immunofluorescence staining assay and some typical clusters of differentiation such as CD34, CD90, CD105 and CD133 by using the cytofluorimetric test assay. RESULTS: Amniotic fluid derived stem cells showed a more primitive phenotype compared to the differentiating potential demonstrated by the other stem cell sources, representing a realistic possibility in the field of regenerative cell therapy suitable for neurodegenerative diseases.

The use of morphokinetic parameters to select all embryos with full capacity to implant.

PURPOSE: Embryo kinetics analysis is an emerging tool for selecting embryo(s) for transfer. The aim of the present study was to determine morphokinetic parameters easily usable in the laboratory and predictive of embryo development and, most importantly, of embryo competence in producing a clinical pregnancy after day 5 transfer. METHODS: A retrospective time-lapse monitoring analysis of morphokinetic parameters for 72 fully implanted embryos (group A) were compared to 106 non-implanted embryos (group B), and to 66 embryos with arrested development from the same pool of group A. All the embryos were from 78 patients undergoing ICSI treatment and day 5 embryo transfers. RESULTS: A day 3 embryo will develop into a viable blastocyst if the following ranges of morphokinetic parameters are met: t1 (between 18.4 h and 30.9 h post-ICSI), t2 (21.4-34.8 h), t4 (33.1-57.2 h), t7 (46.1-82.5 h), t8 (46.4-97.8 h), tC-tF (7.7-22.9 h) and s3 (0.7-30.8 h). On day 5 embryos with the highest probability to implant are those with a cc3 between 9.7 h and 21 h. CONCLUSIONS: Morphokinetic parameters are helpful to make appropriate decisions for the disposition of each embryo. It is recommended that each laboratory should determine its own ranges of in vitro development (IVD-MKP) and implantation-associated (IMP-MKP) morphokinetic parameters.