Semen Examination Research Articles

A novel homozygous mutation in the DNAAF3 gene leads to severe asthenozoospermia and teratospermia.

Primary ciliary dyskinesia (PCD) is an autosomal recessive genetic disorder characterized by ultrastructural defects in the cilia or flagella of cells, causing respiratory abnormalities, sinusitis, visceral transposition, and male infertility. DNAAF3 plays an important role in the assembly and transportation of axonemal dynein complexes in cilia or flagella and has been shown to be associated with PCD. To date, only two cases of PCD with infertility associated with DNAAF3 mutations have been reported, and no mouse models for this gene have been successfully constructed. This study was conducted on an infertile Chinese male patient with a history of bronchitis. Examination of the patient's semen revealed severe asthenozoospermia and teratospermia. Whole exome sequencing revealed a new homozygous loss-of-function DNAAF3 mutation. CRISPR-Cas9 gene-editing technology was used to construct the same mutation in C57/B6 mice, revealing that homozygous C57/B6 mice were characterized by severe hydrocephalus and early death. The results of this study expand the mutation spectrum of DNAAF3 and confirm its correlation with PCD pathogenesis. This study provides new insights on the mechanisms underlying male infertility related to DNAAF3 mutation and PCD.

Analysis of 12 kinds of cytokines in seminal plasma by flow cytometry and their correlations with routine semen parameters

ObjectiveTo investigate the levels of 12 kinds of cytokines in seminal plasma and their correlations with routine semen parameters. MethodsThe remaining seminal plasma samples of 134 patients undergoing routine semen examination were collected for detecting cytokines. The parameters for sperm concentration, percentage of progressively motile sperm (PR), and motility were analyzed by a computer-assisted sperm analysis (CASA) system. According to the results of sperm concentration, PR and motility, 134 patients were divided into the normal routine semen parameters group, oligoasthenospermia group and azoospermia group. The levels of 12 kinds of cytokines in seminal plasma, including interleukin (IL)-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12P70, IL-17, interferin (IFN)-α, IFN-γ, and tumor necrosis factor (TNF)-α, were detected by flow cytometry. Two seminal plasma samples were detected for 10 times, respectively, to calculate the coefficients of variation (CV) of each cytokine. The linear range of each cytokine was measured using the standard, and the correlation coefficient (r) was calculated. ResultsThe r2 of 12 kinds of cytokines detected by flow cytometry were all greater than 0.99. The reproducibility of 2 seminal plasma samples showed that the CVs of all cytokines were lower than 15 % except for TNF-α in sample 1 (15.15 %). Seminal plasma IL-6 levels were negatively correlated with semen volume (P < 0.01). Seminal plasma IL-5 levels were positively correlated with sperm concentration (P < 0.01). Seminal plasma IL-8 levels were negatively correlated with sperm motility (P < 0.01). Seminal plasma IL-8, IL-17 and IL-12P70 levels were negatively correlated with sperm PR (P < 0.05). In addition to the significant negative correlation between IL-5 and IL-17 (P < 0.05), there was a significant positive correlation between the majority of other cytokines. The levels of seminal plasma IL-17 and IL-12P70 in the oligoasthenospermia group and IL-1β and IL-12P70 in the azoospermia group were significantly higher than those in the normal routine semen parameters group (P ≤ 0.05), while the levels of IL-10 in the azoospermia group were significantly lower than that in the normal routine semen parameters group (P < 0.05). ConclusionThere are certain correlations between seminal plasma cytokines and routine semen parameters and strong correlations between different seminal plasma cytokines, suggesting that the imbalance between seminal plasma cytokines may affect sperm quality. However, it still needs to be further confirmed by large samples and multi-center clinical studies and related basic researches.

First-line infertility treatment in normal or subnormal sperm: Interest of a simplified pre-IMSI test.

In the field of male infertility, when sperm is normal/subnormal, a few "add-on" routine tests can complete the basic semen examination. The aim of this study was to develop and evaluate a faster, simplified motile sperm organelle morphology examination (MSOME) technique for selected infertile patients with apparently normal/subnormal sperm and, in their background: failure of two or three intrauterine insemination (IUI) cycles, repeatedly fragmented embryos, embryonic development to blastocyst-stage failures, repeated miscarriages, a long period of infertility or 2 or more IVF attempts without pregnancy. Our test results were correlated with IUI, conventional in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI) and intracytoplasmic morphologically selected sperm injection (IMSI) outcomes. We validated an adapted version of the MSOME analysis called the pre-IMSI test (PIT), based on vacuole evaluation alone. 248 infertile patients from our assisted reproductive technology (ART) Center were retrospectively selected and split into three PIT score subgroups (patients with ≤8% (score I), 9 to 15% (score II) and ≥16% normal spermatozoa (score III)) based on the correlation between PIT results and each ART technique outcome. The choice of one or another of these ART techniques had been made according to the usual clinico-biological criteria. Clinical outcomes for each of the three PIT subgroups were compared individually for the different ART techniques. For ICSI, the effect of the PIT score subgroup was significant for clinical pregnancies (p = 0.0054) and presented a trend for live births (p = 0.0614). Miscarriage rates of IVF attempts were statistically different depending on the PIT score (p = 0.0348). Furthermore, the odds ratios of clinical pregnancy rates were significantly different according to PIT score subgroup when comparing ICSI vs. IMSI or IVF vs. ICSI attempts. IMSI appears to be recommended when sperm belongs to PIT score I, ICSI when it belongs to PIT score II and IVF or IUI when sperm is of PIT score III quality in selected infertile couples. The lack of statistical power in these PIT subgroups means that we must remain cautious in interpreting results. Our results support the interest of this simplified test for certain couples with normal/subnormal sperm to help choose the most efficient ART technique, even as first-line treatment.

P-107 Sperm DNA fragmentation impacts embryonic developmental potential but is not associated with PGT-A outcomes

Abstract Study question Does sperm DNA Fragmentation Index (DFI) affect embryonic development and influence Preimplantation Genetic Testing for Aneuploidy (PGT-A) outcomes in IVF? Summary answer DFI adversely affects early embryonic development, diminishing blastocyst potential. Nonetheless, PGT-A outcomes are unaffected, underscoring the importance of selecting optimal sperm for enhanced IVF results. What is known already Several major factors are thought to cause DFI. The relationship between sperm DFI and IVF results remains debated. Study design, size, duration This study was conducted from January 2018 to March 2023, included 56 married couples undergoing IVF after performing a sperm DFI test. The DFI was assessed at the first semen examination and categorized into three groups: low (≤15%), moderate (15-30%), and high (≥30%). Participants/materials, setting, methods Intracytoplasmic sperm injection (ICSI) was performed on 1006 mature oocytes, followed by blastocyst culture. A total of 278 blastocysts meeting freezing criteria were vitrified. Trophectoderm biopsies from 216 blastocysts underwent Next-Generation Sequencing for aneuploidy analysis. Statistical analysis utilized the Cochran-Armitage test to compare outcomes among the DFI groups. Main results and the role of chance The average age of the husbands and wives who underwent IVF after the DFI examination was 42.3±4.2 and 41.4±2.4 years old. Normal fertility rates after ICSI in low, moderate, and high DFI were 85.5%, 84.5%, and 83.1%, with no significant difference. Similarly, the blastocyst freezing rates were 35.4%, 26.3%, and 29.6%, and tended to decrease as the DFI value increased (p &amp;lt; 0.05). However, the euploid rates for PGT-A were 22.4%, 16.3%, and 18.2%. There were no statistically significant differences between the DFI groups in terms of euploidy. Limitations, reasons for caution Cases declining ICSI due to patient preference or historical medical reasons were excluded from this study. Wider implications of the findings This suggests that sperm DNA imperfections have a negative impact on the developmental process during the early stages of division, but during late stages of division, the ability to repair DNA works to differentiate into blastocysts with normal developmental potential. Trial registration number not applicable

O-018 Utilisation of second successive ejaculate as a way to overcome poor semen parameters for couples who wish to undergo conventional-IVF

Abstract Study question Can a second successive-ejaculate (SSE) be an option for patients with poor sperm parameters (PSPs) on ovum pick-up (OPU)-day who prefer to undergo conventional-IVF (c-IVF)? Summary answer SSE can be a valid option for patients with PSPs who do not wish to undergo ICSI as an assisted reproductive technology (ART) treatment. What is known already ART is applied when first-line treatment for infertility (timed intercourse or intrauterine insemination) is unsuccessful. The male partner is requested to follow ejaculatory abstinence, which according to the WHO guidelines is 2-7 days, while ESHRE recommends 3-4 days. Among these couples there are cases that require ICSI due to semen analysis with PSPs on the day of OPU. However, some couples are reluctant to undergo ICSI and prefer c-IVF as a treatment method. For those cases, we request a second semen sample on the same day (time interval: 1-4 h) and c-IVF is performed using the two ejaculates combined. Study design, size, duration This is a single-centre retrospective study employing 212 semen from male partners with PSPs in the first semen collection on the OPU-day and subsequently, an SSE was requested in order to perform c-IVF according to patients’ wishes. The data were collected from May 2006 to October 2023 in our clinic (SSE-group). The Control group included 271 semen samples from men where c-IVF was conducted using one ejaculate from January to December 2021 in our clinic. Participants/materials, setting, methods The sperm parameters were analysed according to the WHO manual for the examination and processing of human semen and compared between the first and second ejaculates in the SSE-group. Fertilisation rate (FR), percentage of clinically utilised embryos and clinical pregnancy rate were compared between the SSE-group and the Control group. The SSE-group had 254 ART-cycles and the mean maternal age was 36.8±4.9y.o. The Control group included 432 ART-cycles and the mean maternal age was 38.1±5.2y.o. Main results and the role of chance In SSE-group the semen volume of the first ejaculate was significantly higher compared to the second ejaculate (P&amp;lt; 0.0001). The sperm concentration and sperm motility were significantly higher in the second ejaculate compared to the first one (P&amp;lt;0.0001). In the same fashion, the total-motile sperm-count was significantly higher in the second ejaculate compared to the first (P=0.01). All the semen parameters in the Control group were significantly higher compared to both first and second ejaculates from the SSE-group (P&amp;lt;0.00001). The normal FR was significantly higher in the Control group compared to the SSE–group (P&amp;lt;0.01). On the other hand, there was no significant difference in the percentage of embryos that could be utilized (cryopreserved + freshly-transferred) between the two groups (P=0.218). Pregnancy rate for fresh embryo transfers (ETs) was significantly higher in the SSE-group compared to the Control (P=0.005), while there was no significant difference between the two groups for pregnancy rate for frozen/thawed ETs (P=0.728). The main strength of our study is that the utilisation of the SSE combined with the first ejaculate did not decrease the pregnancy rate for the couples with PSPs that wished to follow c-IVF as their treatment and it could actually provide an effective option for them. Limitations, reasons for caution SSE may improve the sperm parameters and give the opportunity to infertile couples to undergo c-IVF according to their wish. However, since the FR was lower in the SSE-group, it is of paramount importance to provide sufficient explanation to those couples and obtain their consent when requesting an SSE. Wider implications of the findings Overall, SSE had positive effect to a large number of parameters evaluated in this study. Additionally, the rate of utilised embryos was similar between the two groups. Two successive-ejaculates combined can be employed without any negative impact for couples with PSPs on the OPU-day that are reluctant to undergo ICSI. Trial registration number not applicable

Investigating the effects of the Omicron variant of COVID-19 on sperm parameters and serum levels of male sexual hormones: Prospective observational study

BackgroundWith the progress and prevalence of COVID-19, concerns have arisen regarding its impact on men's sexual health. Therefore, this study was conducted with the aim of examining the effects of COVID-19 on serum levels of sex hormones and semen. MethodsSixty participants who met the study inclusion criteria enrolled in this study between January and April 2022. The individuals were divided into three groups (n = 20): healthy, COVID-19 positive, and recovered from COVID-19. Blood and semen samples were collected from the participants. Serum levels of sex hormones and semen were evaluated both macroscopically and microscopically. ResultsOur study results showed that the most common symptoms observed in the COVID-19 group were cough (100 %), fever (100 %), fatigue (95 %), and runny nose (90 %). Serum levels of sex hormones (testosterone, FSH, and prolactin) in the COVID-19 group were significantly decreased compared to the healthy group. Microscopic examination of semen revealed significant differences in vitality, progressive, and motile parameters among the three groups, with a decrease observed in the COVID-19 group. ConclusionThese results indicate that COVID-19 may have a negative impact on men's sexual health, potentially affecting hormone production and sperm quality. Further research is needed to determine the long-term effects of COVID-19 on male fertility and to explore potential treatment options.

Abstinence for sperm sample collection and ART outcome: an unsubstantiated myth.

Semen analysis is a basic component of male evaluation. Reproductive centers typically instruct men to provide a semen specimen based on recommendations from WHO Standard for semen examination. Evidence that these recommendations optimize sperm reproductive capacity is lacking. Existing data to optimize sperm quality with shorter abstinence were reviewed. Several recent studies have reviewed the effects of shorter ejaculatory abstinence of semen quality and assisted reproductive technology (ART) outcomes. Shorter abstinence was defined as 1 h-1 day in one review, and <4 h in the other systematic meta-analysis and review. Prior instructions for male patients have not been designed to optimize fertility potential for semen analyses. Optimal sperm quality is obtained by instructing men to have a short abstinence (certainly <1 day, and preferably <4 h) for semen specimens used for in vitro fertilization (assisted reproduction).

A loss-of-function variant in ZCWPW1 causes human male infertility with sperm head defect and high DNA fragmentation.

Male infertility is a global health issue. The more causative genes related to human male infertility should be further explored. The essential role of Zcwpw1 in male mouse fertility has been established and the role of ZCWPW1 in human reproduction needs further investigation to verify. An infertile man with oligoasthenoteratozoospermia phenotype and his parents were recruited from West China Second University Hospital, Sichuan University. A total of 200 healthy Han Chinese volunteers without any evidence of infertility were recruited as normal controls, while an additional 150 infertile individuals were included to assess the prevalence of ZCWPW1 variants in a sporadic male sterile population. The causative gene variant was identified by Whole-exome sequencing and Sanger sequencing. The phenotype of the oligoasthenoteratozoospermia was determined by Papanicolaou staining, immunofluorescence staining and electron microscope. In-vitro experiments, western blot and in-silicon analysis were applied to assess the pathogenicity of the identified variant. Additionally, we examined the influence of the variant on the DNA fragmentation and DNA repair capability by Sperm Chromatin Dispersion and Neutral Comet Assay. The proband exhibits a phenotype of oligoasthenoteratozoospermia, his spermatozoa show head defects by semen examination, Papanicolaou staining and electron microscope assays. Whole-exome sequencing and Sanger sequencing found the proband carries a homozygous ZCWPW1 variant (c.1064C > T, p. P355L). Immunofluorescence analysis shows a significant decrease in ZCWPW1 expression in the proband's sperm. By exogenous expression with ZCWPW1 mutant plasmid in vitro, the obvious declined expression of ZCWPW1 with the mutation is validated in HEK293T. After being treated by hydroxyurea, MUT-ZCWPW1 transfected cells and empty vector transfected cells have a higher level of γ-H2AX, increased tail DNA and reduced H3K9ac level than WT-ZCWPW1 transfected cells. Furthermore, the Sperm Chromatin Dispersion assay revealed the proband's spermatozoa have high DNA fragmentation. It is the first report that a novel homozygous missense mutation in ZCWPW1 caused human male infertility with sperm head defects and high DNA fragmentation. This finding enriches the gene variant spectrum and etiology of oligoasthenoteratozoospermia.

Self-Rated Health and Semen Quality in Men Undergoing Assisted Reproductive Technology

Numerous studies have documented the association of self-rated health (SRH) with chronic diseases. However, few studies have investigated its association with semen quality. To examine the association of SRH with semen quality among men undergoing assisted reproductive technology (ART) in China. This cross-sectional study recruited male partners in couples undergoing ART treatment at the Center for Reproductive Medicine, Tongji Hospital, Wuhan, China. A total of 1262 men underwent 2 semen examinations and completed a questionnaire on SRH between December 2018 and January 2020. Data analysis was performed from November 20, 2022, to March 24, 2023. SRH, including overall physical and mental health, as well as reproductive-related physical and mental health specifically, were reported at baseline recruitment. Sperm concentration, sperm progressive motility, sperm motility, and sperm count as semen quality parameters. The study included 1262 men with a mean (SD) age of 32.79 (5.25) years and body mass index of 24.37 (3.68). Men with poorer SRH had lower semen quality (eg, sperm concentration among poor vs very good overall physical health: percentage variation, -14.67%; 95% CI, -23.62% to -4.66%). Among 4 components of SRH, a greater reduction in semen quality was estimated for reproductive-related SRH compared with overall SRH, whereas the greatest reduction was observed for reproductive-related physical SRH. In comparison with men with very good reproductive-related physical SRH, men with poor reproductive-related physical SRH had differences of -24.78% (95% CI, -32.71% to -15.93%) and -25.61% (95% CI, -33.95% to -16.22%) in sperm count and concentration, respectively, and regression coefficients of -9.38 (95% CI, -12.01 to -6.76) and -9.24 (95% CI, -11.82 to -6.66) for sperm motility and sperm progressive motility, respectively. In this cross-sectional study of Chinese men, poorer SRH was associated with lower semen quality, and reproductive-related physical SRH was the most pronounced indicator. Our findings suggest that SRH, especially reproductive-related physical SRH, was a good indicator of semen quality, which should inform public and clinical regulatory decisions.

Application of contrast-enhanced ultrasound in the diagnosis of tuberous vas deferens tuberculosis.

To assess the value of contrast-enhanced ultrasound (CEUS) in the diagnosis of tuberous vas deferens tuberculosis (VD TB) and improve the positive diagnostic rate of VD TB. CEUS and routine ultrasound (US) images of 17 patients with tuberous VD TB confirmed by surgery, pathology, or laboratory semen examination were retrospectively analyzed and summarized, and the positive rates of both imaging techniques were compared. The 19 VD lesions of the 17 patients were divided into two types according to the CEUS findings: Type I and Type II, and type II was divided into Types IIa, IIb, and IIc. Of the nodules with transverse diameters > 1cm, 100% presented as type II. Of the nodules with transverse diameters < 1cm, 37.5% (3/8) presented as type I and 62.5% (5/8) presented as type II. The sonographic manifestations of tuberous VD TB were hypoechoic and mixed echoic. The positive diagnostic rate was 89.5% for CEUS and 68.4% for US, but the difference was not significant (χ2 = 2.533; P = 0.111). CEUS was able to show the blood supply characteristics of tuberous VD TB, the internal necrosis of nodules was more easily observed by CEUS than by routine US, which is helpful for the diagnosis of tuberous VD TB.

Androloji laboratuvarlarında semen analizi standardizasyonun Dünya Sağlık Örgütü (DSÖ) kriterlerine göre değerlendirilmesi

OBJECTIVE: Due to its complexity and subjectivity, semen analysis is difficult to standardize across laboratories. Our aim in this study is to collect basic information about semen analysis of andrology laboratories in Türkiye and to determine the level of standardization within and between laboratories for semen analysis. MATERIAL and METHODS: A survey consisting of 41 questions covering all aspects of semen analysis was sent to 145 andrology laboratories that perform semen analysis in Türkiye. Of all, 106 laboratories filled out and returned the survey. The content of the survey was prepared according to the procedures specified in the 6th edition of the 2021 World Health Organization (WHO) laboratory handbook for the examination and processing of human semen. The survey was designed to obtain information about the reported parameters, semen collection and storage, macroscopic-microscopic evaluation, advanced tests and internal-external quality control of the laboratory. RESULTS: In relation to the laboratories, 84.1% of the parameters reported in semen analysis, 75.8% of the semen collection phase, 76.5% of the storage of semen in the laboratory and macroscopic analysis, 78.07% of the microscopic analysis of semen, 49.8% of advanced tests and 73% of the parameters related to quality criteria were compatible with WHO 2021 criteria. CONCLUSION: There appear to be differences between laboratories in Türkiye in the reporting and standardization of semen analysis according to the WHO 2021 criteria. Many different standards and criteria are used for semen analysis in andrology laboratories. This makes it difficult to compare results from one laboratory to another. Therefore, it should be recommended that semen analysis be evaluated according to the criteria set by international standards and guidelines. Keywords: semen analysis, andrology laboratory, microscopic analysis

Efficacy of varicocelectomy in improving semen parameters in patients with clinical varicocele

Despite various studies published in the recent years, efficacy of varicocelectomy and its influence on semen parameters, particularly sperm count, motility and morphology has not yet been determined. Conflicting results obtained in different studies, accounts for sparked interest in varicocele management and its effects on seminal parameters. Therefore, present study was designed in an attempt to evaluate the effect of varicocelectomy on the semen parameters by analyzing reports that included men with varicoceles who had both preoperative and postoperative semen examination done. To determine the efficacy of varicocelectomy in improving semen parameters semen parameters (count, motility and morphology) by analyzing reports that included men with varicoceles who had both preoperative and postoperative Spermatogram. The present retrospective study enrolled 50 patients in Tripoli University Hospital medical center, Tripoli /Libya.&#x0D; Varicocele was graded by Dubin grading system by palpation in standing and during Valsalva maneuver under adequate exposure and scrotal Doppler ultrasound Macro and microscopic assessment of semen samples was carried out according to WHO guidelines 2010. The surgical approach used for varicocelectomy was inguinal (low ligation procedure), post operative semen analysis done after 6 months from surgery. Significant improvement in semen parameters (viz sperm counts/ml, sperm counts/ejaculate and sperm motility) were observed after varicocelectomy. We noted no effect of patient’s age on the degree of improvement in semen parameters post operatively. Preoperatively, men with Grade IV varicoceles had lower sperm counts and sperm motility, but significant improvement was observed post-operatively compared to men with Grades II and III varicoceles

#58 : Relationship Between E-Cigarettes (Vaping) Habits and Semen Parameters Among Patient Attending Sabahcare Fertility Centre

Background and Aims: Exposure to nicotine is known to reduce sperm count and motility as well as increasing abnormal parameters. This study aimed to determine the differences of Seminal Fluid Analysis (SFA) between e-cigarettes users (vaping) and non-vaping patients attending SabahCare Fertility Centre. This study is still on-going, more samples are to be expected before the 12[Formula: see text] Congress of ASPIRE Method: Data is collected by handing out questionnaire to male patient doing SFA in the centre. Samples are examined according to WHO laboratory manual for the examination and processing of human semen, sixth edition (WHO, 2021) criteria. Sperm parameters we applied are normozoospermia, oligozoospermia (sperm concentration &lt;14 × 106/ml), azoospermia (no spermatozoa in the ejaculate), asthenozoospermia (sperm motility and/or progressive motility are below the reference value). Chi-Square test analysis will be performed to determine the significance level of the results. Results: Currently, a total of 38 sample sizes are collected. 60.5% are in the normal SFA quality and 39.5% are in the abnormal SFA quality. Conclusion: Sample collection to determine if using e-cigarettes (vaping) affects semen quality among SabahCare Fertility Centre are still ongoing.

Complex genetic and semen examination in patients with the disomy Y

Background. Among the syndromes with aneuploidy on the sex chromosomes (gonosomes), the Y chromosome dysomy presents the mildest phenotypic manifestation. Most 47,XYY patients are not developed reproductive system abnormalities. Some of them have spermatogenesis defects and in-/subfertility, but the prevalence of male infertility in patients with dysomy Y, is not known. The causes and factors of phenotypic variability, fertility disorders, the spermatogenesis and semen characteristics in patients with dysomy Y have not been sufficiently studied.Aim. A comprehensive genetic examination, assessment of the state of spermatogenesis and spermatological defects in patients with dysomy Y.Materials and methods. We examined 25 men with Y-chromosome dysomy. The age of the patients was 32 ± 10 (14–59) years. Chromosome analysis was performed on cultured peripheral blood lymphocytes using a standard cytogenetic method with GTG-staining. Fluorescent in situ hybridization was performed to analyze X and Y chromosomes, gonosomal mosaicism, as well as to evaluate sperm aneuploidy. The Y chromosome microdeletions were detected by multiplex polymerase chain reaction. A standard semen analysis was performed in accordance with the recommendations of the WHO Guidelines (2010). Quantitative karyological analysis of immature germ cells from the ejaculate sediment was performed according to our own previously developed method.Results. Non-mosaic dysomy Y was diagnosed in 22 patients, including 21 men with karyotype 47,XYY, and one patient with additional chromosomal anomality (Robertson translocation) – karyotype 46,XYY,der(13;14)(q10;q10). XYY/XY mosaicism was revealed in two patients, one has a complex Y chromosome mosaicism with a complete the AZFc (b2/b4) deletion. Pathogenic Y chromosome microdeletions were not found in other patients. In one man, the presence of dysomy Y was suspected according to the results of quantitative fluorescent polymerase chain reaction, but according to the results of cytogenetic examination, the presence of an isodicentric chromosome Yq – karyotype 46,X,psu dic(Y)(p11.3) was detected. Using whole exome sequencing in one azoospermic patient, a heterozygous variant c.653G&gt;A(p.Gly218Asp) was detected in the SYCP2 gene, which encodes one of the proteins of the synaptonemal complex.Azoospermia (n = 9, 45 %), oligoastenoteratozoospermia (n = 6, 39 %), asthenoteratozoospermia (n = 3, 17 %), and asthenozoospermia (n = 2, 11 %) were diagnosed among 20 semen analysed patients. The volume of ejaculate was 2.7 ± 1.7 (1.0–5.0) ml, the concentration and total number of spermatozoa was 14.9 ± 21.8 (0.0–66.25), 50.4 ± 77.7 (0.0–265.0) mil lions, respectively. Oligospermia was observed in 2 (10%) patients. The quantitative karyological analysis of immature germ cells allowed to detect cryptozoospermia in azoospermic patinents and defects of prophase I of meiosis.Fluorescent in situ hybridization analysis revealed increased sperm aneuploidy (gonosomal dysomy and nullisomy, dysomy 18 and 21).Conclusion. Most of infertile men with dysomy Y are non-mosaic, and characterized by defected spermatogenesis (non-obstructive azoospermia or oligozoospermia), due to meiotic arrest at prophase I. Pathogenic AZFc deletion was found in a patient with complex Y chromosome mosaicism. Additional genetic factors of male fertility and spermatogenesis defects were found in some patients, which may explain the phenotypic variability.

Detection of Melanocortin Receptor Type 4 (MC4R) Gene in Semen of Etawah Crossbreed and Senduro Goats

Etawah crossbreed and Senduro goats as superior local goats and biological genetic resources that must be preserved in Indonesia. Melanocortin receptor type 4 (MC4R) is the primary gene that regulates food intake and energy balance which can be used as a candidate marker for livestock selection genes. This study aimed to analyze the comparison of cement quality and detect the profile of the MC4R gene in Etawah crossbreed and Senduro goat semen. The samples of this study were Etawah crossbreed and Senduro goat semen. Macroscopic examination of cement includes the evaluation of volume, consistency, odor, color, and pH. Microscopic examination of semen includes motility, abnormalities, and spermatozoa concentration. Examination of motility was performed in the condition of fresh semen, after the addition of diluents, and post-thawing of frozen straw. The polymerase chain reaction (PCR) method was performed to amplify specific fragments of the MC4R gene. There was a decrease in spermatozoa motility before freezing and post-thawing frozen semen compared to fresh semen. The quality of frozen straw post-thawing Etawah crossbreed and Senduro goat was still worth using for artificial insemination and in vitro embryo production. The MC4R gene can be detected in liquid and frozen semen from Etawah crossbreed and Senduro goats using a PCR test in the form of a single band along 642 bp.

Varicocelectomy in Male Subfertility Treatment: ‘‘Updated’’ Indications for Surgery

We analyzed the recent literature data on varicocelectomy in treatment men from infertile couples. There are still a lot of unresolved issues that require clarification. It is still unclear the exact pathogenesis of testicular malfunction in some men with varicocele. Recent European and American urological guidelines recommend varicocele repair in men from infertile couples with “abnormal” semen parameters. Although, according to the 6th ed. of WHO laboratory manual for the examination and processing of human semen it is not clear the cut-off values between normal and abnormal semen parameters. Also, still unclear why approximately third part of patients do not experience any improvement after varicocele repair. According to the recent data analysis, total progressively motile sperm count (TMSC) and sperm concentration can be the significant predictors of semen improvement and pregnancy after varicocele repair. However, it should be remarked that the mean quality of existed data from low to moderate. All mentioned issues indicate the necessity of further large-scale multicenter randomized clinical trials.

Improving standard practices in studies using results from basic human semen examination.

The purpose of this article is to provide an explanation of the background behind a checklist that declares the laboratory methods used in a scientific study. It focuses primarily on implementing laboratory procedures to yield reliable results in basic semen examinations. While the World Health Organization (WHO) and international standards provide recommendations for basic semen examination, manuscripts submitted to Andrology frequently lack transparency regarding the specific techniques used. In addition, the terminology used for semen examination results often fails to provide a clear definition of the groups under study. Furthermore, the WHO's reference limits are often misinterpreted as strict boundaries between fertility and infertility. It is important to note that valid clinical andrological diagnoses and treatments cannot rely solely on semen examination results; they require proper laboratory procedures as a foundation for diagnosing and treating male patients. Therefore, scientific journals should promote the adoption of robust laboratory practices and an accurate definition of patient groups. A checklist can facilitate the design of high-quality studies and the creation of informative publications. Further, it can help journals assess submitted manuscripts and improve the overall quality of their publications.

O-285 The 6th edition of the WHO manual for the examination and processing of human semen: How should it affect our practice?

Abstract The WHO laboratory manual (World Health Organization 2021) has widened the scope of semen examination from primarily a prognostic tool for Medically Assisted Reproduction (MAR) by indicating the importance of evaluating men with possible disorders in male reproductive functions. Practical instructions for basic examination have been clarified and focused to measures of sperm production and motility as judged from sperm number, motility (vitality if poor motility) and sperm morphology. In addition to the recommendations by the WHO, an international standard based on the same laboratory science as the WHO manual has been published (International Organization for Standardization 2021) to facilitate for laboratories to provide reliable results of semen examination. Each ejaculate must be handled with great care before analysis begins. Time and temperature between sample collection and start of assessments is crucial. To reduce assessment variability, it is essential to ascertain that the examined aliquots are representative for the entire ejaculate and that the number of observations is sufficient to reduce the risk for significant influence of random factors. The former is handled both by aliquot volume (at least 50 µL for sperm concentration) and replicate assessment and comparison that the two assessments don’t differ too much. Regarding number of observations, 400 sperm counted in sperm concentration and in sperm motility assessments is required to reduce this source of errors to ± 10%. For assessment of sperm morphology and vitality a minimum number of observations is 200 spermatozoa. To achieve acceptable performance, thorough in-house training is necessary. There are protocols how to set up and run such schemes (Mortimer 1994, 1994). Basic medical laboratory standards also require that the laboratory regularly performs internal quality control to monitor that inter- and intra-personal variability is under control. Each laboratory should also participate in an external scheme for quality assessment (EQA) (International Organization for Standardization 2012). For the proper interpretation of semen examination results reference limits are important. However, the reference limits suggested by the WHO (World Health Organization 2021) come from a very mixed group of men and should therefore not be mistaken for true limits between fertility and infertility. The new WHO manual therefore argues for development of decision limits (when is it reasonable to act?) that are much more important than limits from a mixed population. It has been argued that semen examination must be better standardized (Björndahl et al. 2016), but the compliance has been almost non-existent (Vasconcelos et al. 2022) leading to even stronger appeals for stronger improvement of basic laboratory investigations of male reproductive functions (Björndahl et al. 2022) References Björndahl L, Barratt CL, Mortimer D, and Jouannet P. ‘How to count sperm properly': checklist for acceptability of studies based on human semen analysis. Hum Reprod 2016: 31; 227-232. Björndahl L, Barratt CLR, Mortimer D, Agarwal A, Aitken RJ, Alvarez JG, Aneck-Hahn N, Arver S, Baldi E, Bassas L, et al. Standards in semen examination: publishing reproducible and reliable data based on high-quality methodology. Hum Reprod 2022. International Organization for Standardization. ISO 15189:2012 Medical Laboratories – Requirements for Quality and Competence 2012. Geneva. International Organization for Standardization. ISO 23162:2021 Basic semen examination — Specification and test methods 2021. ISO, Geneva. Mortimer D. Laboratory standards in routine clinical andrology. Reproductive Medicine Review 1994: 3; 97-111. Mortimer D. Practical Laboratory Andrology 1994. Oxford University Press, Oxford. Vasconcelos AL, Campbell MJ, Barratt CLR, and Gellatly SA. Do studies published in two leading reproduction journals between 2011 and 2020 demonstrate that they followed WHO5 recommendations for basic semen analysis? Hum Reprod 2022. World Health Organization. WHO laboratory manual for the examination and processing of human semen. 6th edn, 2021. World Health Organization, Geneva.

(180) Effects of Electromagnetic Radiation Emittted by Smartphones on Sperm Quality: An in Vitro Study

Abstract Introduction In modern times, the widespread use of mobile phones is a notable source of radiofrequency–electromagnetic radiation (RF-EMR) with a potential impact on human health. The advancements in technologies such as Bluetooth earphones have increased the time the cell phone resides inside the pant pockets of men, in close contact with their testicles. As a result, it makes the testicles more prone to RF-EMR exposure for longer. Recent literature has shown that such long exposure is likely to cause damage to the sperm quality and functioning in several ways, including the increase in local body temperature, high oxidative stress, reactive oxygen species, and free radical formation (1,2). However, the causing effect of modern cell phones and wireless communication networks (4G and 5G) on sperm quality has not been explored. Objective The aim of our study was to investigate the effect of cell phone radiations emitted through communication technologies - 4G, 5G and WIFI on sperm motility and viability using an in-vitro approach and further try to shed light if physical barriers or distance ease these effects. Methods Normozoospermic men (n=18) aged 25-35 were recruited whose samples after semen analysis were divided into three parts i) in close contact with sample without cover ii) mobile phone covered with physical barrier, and iii) phone kept at distance from the samples. Standard semen analysis was carried according to WHO laboratory manual for the examination and processing of human semen, 5th ed. Voice calls over the internet were made using 4G, 5G, or WIFI and using these, RF-EMF exposure was given for six hours which was measured by RF-EMR detection meter. In addition, to check for the heating effect, samples were kept inside the incubator at 37°C for samples exposed to WIFI effects. Continuous variables were presented as medians and interquartile ranges [25th - 75th], and comparison between groups was performed using the U Mann Whitney test. SPSS v.28 was used for statistical analysis. Results A decrease in both parameters under study was evident with WIFI (Motility (50% vs. 38%, p = 0.024; viability 60% vs 47%, p = 0.003), while no decrease was noted with 4G or 5G RF-EMR. Samples connected with WIFI were warmer but samples inside the incubator, when observed had decreased progressive motility (40% vs 24%, p= 0.04). On the other hand, the effect of RF-EMR by WIFI on smartphone was mitigated both for sperm motility and viability (p = 0.01, p = 0.01), n = 18) with the use of a case and on increasing the distance of the contact respectively. Conclusions The result from the study suggests that the radiation emitted by WIFI do have an adverse impact on the motility and viability of sperm. It seems plausible that the effect maybe a result of excess heat generation which was most evident with mobile phones connected to WIFI. However, further studies utilizing different phones and different time periods of exposure are warranted to effectively conclude the findings Disclosure No

Medicolegal Aspects and Sperm Examination Procedures in Rape Cases: A Narrative Literature Review

Sexual violence is usually a covert case where the witnesses are the victim and the perpetrator. For various reasons, even victims may not be able to provide complete information about the perpetrator or the identity of the rapist. With limited initial information, physical and biological evidence found from victims, crime scenes, and perpetrators will play an important role in the objective and scientific reconstruction of the incident. This literature review aimed to describe various sperm examination methods and medicolegal aspects of rape cases. Identification of one or more intact spermatozoa is conclusive evidence of the presence of semen, further confirming sexual contact. The conditions and the sperm collection area play an important role in determining the time interval between deposition and sample collection. Examination of spermatozoa can be done without staining and with staining. Examination of semen can be done by examination (tactile, visual, UV light, acid phosphatase reagent, zinc spot test, and baecchi stain).