The development of new biomarkers of spermatozoa quality in cattle.

There is a current need for new biomarkers of spermatozoa quality, that consistently and correctly identify spermatozoa that will successfully contribute to subsequent embryo development. This could improve the standardization of semen analysis, decrease early embryo mortality, and use these biomarkers as a selection tool before servicing females. This study utilized imaging techniques to identify potential biomarkers of sperm quality, using sires previously classified as high (n = 4) or low (n = 4) performing at producing blastocysts in vitro. Spermatozoa were assessed before and following a gradient purification protocol, to understand how populations of cells are impacted by such protocols and may differ between in vivo and in vitro use. Pre-gradient samples from low-performing sires had an increased incidence of DNA damage, although post-gradient samples from high-performing sires were found to have an increased incidence of DNA damage. When evaluating morphology via fluorescent microscopy, the most prevalent defects in pre-gradient samples from high-performing sires were tail defects, which are successfully removed during purification processing. The most prevalent defects in pre-gradient samples from low-performing sires were aggresome defects located in the head, which would be brought into an oocyte upon fertilization and may impair embryo development. Image-based flow cytometry (IBFC) was employed to quantify defect prevalence to evaluate a greater sample size decreasing the variability that exists in manual assessments. Using IBFC, aggresome defects were again identified in the heads of spermatozoa from low-performing sires. Post-gradient samples from low-performing sires had a significantly greater (p < 0.05) incidence of aggresome defects than post-gradient samples from high-performing sires. Additionally, IBFC was used to evaluate spermatozoa viability following gradient purification. Distinct populations of sperm cells were identified. High-performing sires had more spermatozoa in the population deemed most viable than low-performing sires. This study demonstrated that spermatozoa defects vary in populations before and following gradient purification, indicating that it may be beneficial to separately evaluate semen for in vivo and in vitro use. Furthermore, a prevalent defect in low-performing sires that could explain a discrepancy between successful fertilization and embryo development was identified. Therefore, elucidating a malfunction regulated by sire, that could potentially affect early embryo development.

The impact of different sperm preparation methods on clinical pregnancy and live birth rates in intrauterine insemination cycles: a retrospective single-center cohort study.

OBJECTIVE: To assess the impact of 2 different sperm preparation methods, density gradient centrifugation and simple wash, on clinical pregnancy and live birth rates in intrauterine insemination (IUI) cycles with and without ovulation induction. DESIGN: Retrospective single-center cohort study. SETTING: Academic fertility center. PATIENTS: In total, 1,503 women of all diagnoses sought IUI with fresh-ejaculated sperm. EXPOSURE: Cycles were divided into 2 groups on the basis of sperm preparation technique: density gradient centrifugation (n = 1,687, unexposed group) and simple wash (n = 1,691, exposed group). MAIN OUTCOME MEASURES: Primary outcome measures consisted of clinical pregnancy and live birth rates. Furthermore, adjusted odds ratios and 95% confidence intervals for each outcome were calculated and compared between the 2 sperm preparation groups. RESULTS: Odds ratios did not differ between density gradient centrifugation and simple wash groups for clinical pregnancy and live birth (1.10 [0.67-1.83] and 1.08 [0.85-1.37], respectively). Additionally, when cycles were stratified using ovulation induction rather than adjusted for, no differences in clinical pregnancy and live birth odds were noted between sperm preparation groups (gonadotropins: 0.93 [0.49-1.77] and 1.03 [0.75-1.41]; oral agents: 1.78 [0.68-4.61] and 1.05 [0.72-1.53]; unassisted: 0.08 [0.001-6.84] and 2.52 [0.63-10.00], respectively). Furthermore, no difference was seen in clinical pregnancy or live birth when cycles were stratified using sperm score or when the analysis was limited to first cycles only. CONCLUSION: Overall, no difference was noted in clinical pregnancy or live birth rates between patients who received simple wash vs. density gradient-prepared sperm, suggesting similar clinical efficacy between the 2 techniques for IUI. Because the simple wash technique is more time-efficient and cost-effective compared with the density gradient, adoption of this technique could lead to comparable clinical pregnancy and live birth rates for IUI cycles, although optimizing teamwork flow and coordination of care.

A short versus a long time interval between semen collection and intrauterine insemination: a randomized controlled clinical trial.

STUDY QUESTION: Does a short interval (i.e. ≤90 min), compared to a long interval (i.e. ≥180 min), between semen collection and intrauterine insemination (IUI) increase the cumulative chance of an ongoing pregnancy after six IUI cycles? SUMMARY ANSWER: A long interval between semen collection and IUI resulted in a borderline significant improvement in cumulative ongoing pregnancies and a statistically significant shorter time to pregnancy. WHAT IS KNOWN ALREADY: Retrospective studies assessing the effect of the time interval between semen collection and IUI on pregnancy outcomes have shown inconclusive results. Some studies have indicated a beneficial effect of a short interval between semen collection and IUI on IUI outcomes, while others have not found any differences. To date, no prospective trials have been published on this subject. STUDY DESIGN, SIZE, DURATION: The study was performed as a non-blinded, single-center RCT with 297 couples undergoing IUI treatment in a natural or stimulated cycle. The study was conducted between February 2012 and December 2018. PARTICIPANTS/MATERIALS, SETTING, METHODS: Couples with unexplained or mild male subfertility and an indication for IUI were randomly assigned for up to six IUI cycles into either the control group (long interval, i.e. 180 min or more between semen collection and insemination) or the study group (short interval, i.e. insemination as soon as possible after semen processing and within 90 min of semen collection). The study was carried out in an academic hospital-based IVF center in the Netherlands. The primary endpoint of the study was ongoing pregnancy rate per couple, defined as a viable intrauterine pregnancy at 10 weeks after insemination. MAIN RESULTS AND THE ROLE OF CHANCE: In the short interval group, 142 couples were analyzed versus 138 couples in the long interval group. In the intention-to-treat (ITT) analysis, the cumulative ongoing pregnancy rate was significantly higher in the long interval group (71/138; 51.4%) compared to that in the short interval group (56/142; 39.4%; relative risks 0.77; 95% CI 0.59-0.99; P = 0.044). The time to pregnancy was significantly shorter in the long interval group (log-rank test, P = 0.012). A Cox regression analysis showed similar results (adjusted hazard ratio 1.528, 95% CI 1.074-2.174, P = 0.019). LIMITATIONS, REASONS FOR CAUTION: Limitations of our study are the non-blinded design, the long inclusion and follow-up period of nearly seven years and the large number of protocol violations, especially because they predominantly occurred in the short interval group. The non-significant results in the per-protocol (PP) analyses and the weaknesses of the study should be taken into account in the assessment of the borderline significance of the results in the ITT analyses. WIDER IMPLICATIONS OF THE FINDINGS: Because it is not necessary to perform the IUI immediately after semen processing, there can be more time available to choose the optimum work-flow and clinic occupancy. Clinics and laboratories should find their optimal timing of insemination, considering the time between human chorionic gonadotropin injection and insemination in relation to the sperm preparation techniques used as well as the storage time and conditions until insemination. STUDY FUNDING/COMPETING INTEREST(S): There were no external funding and no competing interests to declare. TRIAL REGISTRATION NUMBER: Dutch trial registry, trial registration number NTR3144. TRIAL REGISTRATION DATE: 14 November 2011. DATE OF FIRST PATIENT'S ENROLLMENT: 5 February 2012.

Assessment of semen quality and anti-oxidative enzyme activity between bovine sex-sorted and non-sex-sorted frozen-thawed semen.

In the current study, the difference between the sex-sorted and non-sex-sorted frozen semen of Holstein Friesian breed cattle was investigated. Significant variation (p < .05) was found in the semen quality parameters such as motility; vitality; acrosome integrity rate; the anti-oxidative enzyme activity including GSH (glutathione); SOD (superoxide dismutase); CAT (catalase); GSH-Px (glutathione peroxidase) and the rate of fertilization. The results showed that the sperm acrosome integrity and motility of the non-sorted sperm were higher compared to sex-sorted sperm (p < .05). The linearity index and mean coefficient analysis revealed that the percentage of 'grade a' in sex-sorted sperm were significantly (p < .05) lower than non-sorted sperm. Interestingly, low SOD level and high CAT level was found in the non-sexed semen than in the sexed semen (p < .05). Furthermore, the GSH and GSH-Px activity in the sexed semen was found lower than the non-sexed semen (p < .05). In conclusion, sperm motility characteristics were lower in sex-sorted semen than in non-sex-sorted semen. This might be related to the complex process of sexed semen production, which could reduce sperm motility and movement characteristics, acrosomal integrity, CAT, SOD, GSH and GSH-Px, and finally lead to the decline in the fertilization rate.

Liquid storage of Ostrich (Struthio camelus) semen at 5 °C through intermediate dilution.

Dilution rate, dilution temperature and storage time have been recognized as vital steps in the processing of semen for storage before artificial insemination. The objective of this study was to determine optimal dilution and dilution temperature with an ostrich-specific semen extender for chilled storage. Four preselected ostrich (Struthio camelus var. domesticus) males, known for their ease of collection and specific semen quality parameters, were collected using the "dummy" female method. Dilution of 384 semen samples, at rates of 1:1, 1:2, 1:4 and 1:8 semen/diluent ratio with a diluent set at 5, 21 and 38 °C was performed and stored for 48 h at 5 °C. In vitro sperm function tests were conducted to evaluate treated semen during different storage intervals of 1, 5, 24 and 48 h. Motility and kinematic parameters were measured by the Sperm Class Analyzer®, the percentage live sperm measured by fluorescence SYBR14®/PI (LIVE/DEAD®), the percentage of sperm able to resist the hypo-osmotic swelling (HOS) stress test and sperm morphology determined by Nigrosin-Eosin staining. Progressive motility (PMOT), motility (MOT), sperm kinematics, LIVE and HOS were best (P < 0.05) maintained at a higher dilution of 1:4-1:8. The beneficial effect (P < 0.05) of a higher dilution temperature (21 °C) was prominent in terms of PMOT at a higher dilution. Storage of chilled semen at 5 °C requires dilution, at interpolated rates of 1:6-1:7, together with an extender temperature of 21 °C, to maintain optimal sperm function with minimal loss over a 48 h storage period.

A comprehensive evaluation of pre- and post-processing sperm parameters for predicting successful pregnancy rate following intrauterine insemination with the husband's sperms.

BACKGROUND: To determine the predictive values of sperm parameters pre- and post-processing by density gradient centrifugation for clinical pregnancy rates (CPRs) following artificial insemination by husband (AIH) in infertile Chinese couples. METHODS: A total of 3,522 AIH cycles from 1,918 couples were retrospectively analyzed. The parameters were compared between the pregnant and non-pregnant groups and further between different etiological groups (Male-factor, Both-male-and-female-factor, and Other-factor). Multivariate logistic regression analysis was performed to create models for predicting the CPRs of each etiological group. RESULTS: The overall CPR was 13.3%. There were significant improvements for most sperm parameters after DGC. Multivariate logistic regression analysis indicated that, in overall AIH cases, the top parameters significantly influencing the CPR of AIH were pre-STR (OR = 1.037; P = 0.048) and post-VSL (OR = 1.036; P = 0.011). In the Male-factor Group, the top influencing parameters were pre-VCL (OR = 2.096; P = 0.008), pre-LIN (OR = 1.930; P = 0.002) and post-VSL (OR = 1.316; P = 0.023). In the Both-factor Group, the top influencing parameters were pre-VCL (OR = 1.451; P = 0.008) and post-motility (OR = 1.218; P = 0.049). In the Other-factor Group, the top influencing parameters were pre-VAP (OR = 1.715; P = 0.024), pre-STR (OR = 1.20; P = 0.011) and post-VSL (OR = 1.04; P = 0.017). Moreover, receiver operating characteristic analysis showed that the logistic regression models of the Male- and Both-factor Groups had greater powers for prognostic classification than those of other groups. CONCLUSIONS: This study demonstrated that some sperm parameters have a collinearity relationship in predicting the CPR following AIH. Moreover, the predictive capacity of a multivariate logistic regression model is better than those of individual parameters, especially for the Male- and Both-factor Groups. In these cases, pre-VCL is the common top influencing factor.

Retrospective comparison of the semen preparation techniques for intrauterine insemination: Swim-up versus density gradient method.

OBJECTIVE: Our study aims to retrospectively examine the relationship between two different sperm preparation methods used in IUI among eight years in terms of pregnancy and live birth rates. METHODS: We evaluated the data of semen samples between December 2012 and March 2020. Three hundred eighty-four samples prepared with Conventional Swim-up (CSW) and 361 samples prepared with Density Gradient-Swim up (DGC-SW) obtained from men applying for IUI were analyzed. Spermiogram results of the semen samples given by men applying for IUI were examined. Data about sperm preparation method, post washed sperm parameters, pregnancy, and live birth rate were collected. Statistical analysis was performed. RESULTS: Basal progressive sperm count was significantly higher in pregnant couples in both CSW and DGC-SW groups (p = 0,032, p = 0,035, respectively). In each group, the post washed total progressive motile sperm count obtained by CSW and DGC-SW methods were significantly higher in pregnant patients (p < 0.05). There was no significant difference between CSW and DGC-SW methods in pregnancy achievement (p = 0,399, χ2 = 0,712). Live birth and miscarriage rates were not different between the groups (p = 0,243, χ2 = 2.827). CONCLUSION: In conclusion, there is no significant difference between CSW and DGC-SW for pregnancy and live birth rates. Our results suggest that both sperm preparation techniques used in IUI are not superior to each other. In other words, the choice of sperm preparation method does not affect the pregnancy rate in couples undergoing IUI.

The effect of semen collection location and time to processing on sperm parameters and early IVF/ICSI outcomes.

PURPOSE: We aimed to assess whether home collection and increased time to semen processing are associated with altered sperm parameters, fertilization rates (FR), day 5 usable quality blastocyst development rates (D5-UQBR), or pregnancy rates (PR) in patients undergoing IVF/ICSI. METHODS: This was a retrospective cohort study of patients undergoing IVF/ICSI before the coronavirus disease 2019 (COVID-19) pandemic ("clinic" collection, n = 119) and after COVID-19 ("home" collection, n = 125) at an academic fertility practice. Home collection occurred within 2 h of semen processing. Patient sperm parameters, FR (#2PN/MII), D5-UQBR (# transferable and freezable quality blastocysts/# 2PN), and PR in fresh transfer cycles were compared between clinic and home groups with t-tests. The association between time to processing on outcomes was assessed with regression modeling, controlling for potential confounders. RESULTS: Mean male age was 37.9 years in the clinic group and 37.2 years in the home group (p = 0.380). On average, men were abstinent for 3.0 days (SD 1.7) in the clinic group and 4.1 days (SD 5.4) in the home group (p = 0.028). Mean time to semen processing was 35.7 min (SD 9.4) in the clinic group and 82.6 min (SD 33.8) in the home group (p < 0.001). There was no association between collection location and increased time to processing on sperm motility, total motile count, FR, D5-UQBR, or PR. CONCLUSIONS: Our data suggest that increased time to processing up to 2 h with home semen collection does not negatively impact sperm parameters or early IVF/ICSI outcomes.

Characterization of Boar Studs in Brazil / Caracterização das centrais de coleta e processamento de sêmen no Brasil

ABSTRACT: Understanding the flow of processes in swine production systems and how they work is fundamental to improving reproductive performance. We surveyed 32 boar studs in Brazil, representing 61.53% of the total in the country. Commercial lines were the most common breed (59.38%) used in the studs. Individual pens and stalls were the most commonly used as housing system (71.9%), and 81.25% of the studs reported having some form of thermal control system. For most of the studs (62.51%), average weekly sperm concentration was higher than 200 million cells per mL. Also, for most studs (71.88%) average weekly ejaculate volume was more than 250 mL. In 46.88% of the studs each ejaculate yielded 26 to 40 semen doses. In 6.25% of the studs, 3.5 billion sperm cells per dose were used for artificial, intracervical insemination. Sperm concentration in 46.88% of the studs was determined using a computer-assisted system analysis. The assessment of sperm quality was conducted using morphology, concentration, and microbiological testing. Employees working on semen collection had formal education of elementary/middle school (34.38%) and high school (37.5%). Most of the laboratory technicians had frequented high school (75%). The most time-consuming task was semen processing, taking 16 to 25 h a week (46.8%) and 6 to 10 h was allotted for the cleaning of stud facilities (46.8%). The data collected in the present study allow greater knowledge of this important part in the pig production chain in Brazil.

RESUMO: O entendimento dos processos na suinocultura e como estes funcionam é fundamental para o incremento da performance reprodutiva. Por via eletrônica, foi realizada uma investigação com 32 centrais de coleta e processamento de sêmen no Brasil, representando 61,53% do total no país. O número médio de reprodutores por central foi de 122. Reprodutores da linhagem comercial foram os mais frequentemente (59,38%) utilizados nas centrais. As baias para alojamento individual dos reprodutores foram as mais comuns (71,9%) e 81,25% das centrais relataram possuir algum sistema de climatização. Na maioria das centrais (62,51%), a média da concentração espermática foi maior que 200 milhões de espermatozoides/mL. Da mesma forma, a maioria das centrais (71,88%) apresentaram um volume do ejaculado superior a 250 mL. Em 46,88% das centrais um ejaculado produziu de 26 a 40 doses inseminantes. Em 6,25% das centrais eram utilizadas uma concentração de 3,5 bilhões de espermatozoides por dose inseminante na inseminação intracervical. A concentração espermática em 46,88% das centrais era determinada através do sistema CASA. A avaliação da qualidade espermática era realizada através da análise de morfologia, concentração e exame microbiológico das doses inseminantes. Os colaboradores envolvidos com a coleta de sêmen tinham nível de instrução fundamental (43,38%) e médio (37,5%). A maioria dos técnicos no laboratório das centrais tinham ensino médio (75%). A atividade que mais consumia tempo foi o processamento do sêmen, com 16 a 25 horas/semana (46,8%) e 6 a 10 horas era utilizada na limpeza das instalações (46,8%). Os dados coletados no presente estudo permitem um maior conhecimento desse elo da cadeia produtiva da suinocultura no Brasil.

Longer time interval between semen processing and intrauterine insemination does not affect pregnancy outcome.

OBJECTIVE: To study whether the pregnancy outcome of intrauterine insemination (IUI) is affected by a longer time interval between semen processing and insemination. DESIGN: Retrospective cohort. SETTING: Teaching hospital. PATIENT(S): Couples with subfertility and an indication for IUI over a 10-year period. INTERVENTION (S): Insemination performed the day after but within 24 hours of semen collection and processing (delayed insemination) compared with insemination performed immediately after sperm collection and processing (immediate insemination). MAIN OUTCOME MEASURE(S): Ongoing pregnancy rate, defined as a pregnancy confirmed by ultrasound at 10 to 12 weeks of gestation. RESULT(S): In total, 1,136 cycles were analyzed. In 77 of 547 couples (14%) an ongoing pregnancy occurred after delayed insemination, and in 77 of 589 couples (13%) an ongoing pregnancy occurred after immediate insemination. Both groups had similar baseline characteristics. After adjustment for confounders, there was no difference in the ongoing pregnancy rate between delayed as compared with immediate insemination (odds ratio 0.89; 95% confidence interval, 0.63-1.25). CONCLUSION(S): There is no negative effect on pregnancy rate when IUI of processed sperm is delayed until the next day. This approach allows additional flexibility for couples when the male partner is not available on the day of ovulation, and it allows for a spread of workload in the laboratory.

Comparison of two sperm processing techniques for low complexity assisted fertilization: sperm washing followed by swim-up and discontinuous density gradient centrifugation.

OBJECTIVE: This study aimed to assess and compare sperm motility, concentration, and morphology recovery rates, before and after processing through sperm washing followed by swim-up or discontinuous density gradient centrifugation in normospermic individuals. METHODS: Fifty-eight semen samples were used in double intrauterine insemination procedures; 17 samples (group 1) were prepared with sperm washing followed by swim-up, and 41 (group 2) by discontinuous density gradient centrifugation. This prospective non-randomized study assessed seminal parameters before and after semen processing. A dependent t-test was used for the same technique to analyze seminal parameters before and after semen processing; an independent t-test was used to compare the results before and after processing for both techniques. RESULTS: The two techniques produced decreases in sample concentration (sperm washing followed by swim-up: P<0.000006; discontinuous density gradient centrifugation: P=0.008457) and increases in motility and normal morphology sperm rates after processing. The difference in sperm motility between the two techniques was not statistically significant. Sperm washing followed by swim-up had better morphology recovery rates than discontinuous density gradient centrifugation (P=0.0095); and the density gradient group had better concentration recovery rates than the swim-up group (P=0.0027). CONCLUSION: The two methods successfully recovered the minimum sperm values needed to perform intrauterine insemination. Sperm washing followed by swim-up is indicated for semen with high sperm concentration and better morphology recovery rates. Discontinuous density gradient centrifugation produced improved concentration recovery rates.

Processing, selecting and ritualizing: ambivalent relationships to semen.

Two articles on human immunodeficiency virus (HIV) and reproduction have recently been published in Reproductive BioMedicine Online, both describing developments that increase reproductive options for HIV-positive men. A study of a semen-processing technique used at a South African hospital found that two out of 103 processed samples tested positive for HIV DNA and none for RNA, indicating 98.1% and 100% effectiveness, respectively. The authors recommend semen processing followed by viral validation of processed sperm samples when providing assisted reproduction treatment to couples with an HIV-positive male partner. The other article reviews developments such as semen processing, antiretroviral (ARV) therapy and pre-exposure prophylaxis (PrEP), which have all reduced the risk of HIV transmission in the context of reproduction. The author also notes, however, that research on fertility in the context of HIV focuses almost exclusively on heterosexual couples, and has overlooked the links between reproduction, HIV and homosexuality. This article analyses the ambivalent role of semen - associated with both reproduction and infection - and how reproductive medicine and health care in different ways seek to 'get hold' of sperm. By taking this analytic approach, sex and parenthood can be thought of as two different but related kinds of intimacy and kinship.

Semen decontamination for the elimination of seminal HIV-1.

The risk of human immunodeficiency virus (HIV) transmission to the female partner, or potential offspring of an HIV-1 infected man can be reduced using semen decontamination procedures before assisted reproductive treatment (ART). The objective of this study was to determine the efficiency of decontaminating semen samples (n = 186) from 95 HIV-1 sero-positive patients. Aliquots of neat semen were submitted for viral validation by qualitative and quantitative polymerase chain reaction. Semen samples were processed by density gradient centrifugation in combination with a ProInsert™ tube after which aliquots of the processed sperm samples were analysed for the presence of HIV-1. Fifty-four percent of all tested neat semen samples tested positive for HIV-1 DNA, RNA or both (13.4%, 11.3% and 29.0%, respectively). From a total of 103 processed sperm samples that were submitted for viral validation, two samples tested positive for HIV-1 DNA and none for RNA. In conclusion, semen processing with the ProInsert™ followed by viral validation of processed sperm samples should be carried out when providing ART to couples where the male partner is HIV-1 sero-positive.

Identification and preparation of sperm for ART.

State-of-the-art techniques attempt to select sperm with the best functional capacity to produce pregnancy and, subsequently, healthy offspring. A variety of approaches are now being evaluated. Future approaches may allow for selection of sperm based on sperm DNA integrity, degree of aneuploidy, or apoptosis. Other approaches involve attempting to improve the in vitro function of sperm with exposure to compounds such as pentoxifylline or platelet activating factor. In the future, we are likely to see significant improvements in the ability to select the best sperm for assisted-reproductive-technology procedures and the use of these procedures in routine clinical practice.

Elimination of HIV-1 from semen and application of the processed semen to assisted reproductive technology.

As HIV infection is becoming a controllable chronic infection after the introduction of the highly active antiretroviral therapy (HAART), patients with this infection are now able to make plans for the future. Numerous serodiscordant (HIV-positive men and HIV-negative women) couples seek medical assistance to have a child with minimum risk to the HIV-negative female partner and the child using processed (washed) sperm. HIV-1 elimination from the semen using density gradient centrifugation and the swim-up procedure, which is a standard processing procedure, depends on both sperm motility and the physical properties of the virus-containing particles. To establish a simple method for recovering sperm from poor quality semen, we developed the tilted-tube rotation method to generate a continuous density gradient, and using this continuous density gradient we have succeeded in recovering motile sperm, even from the semen of seropositive males with severe male factors. This review aims to summarize the recent progress in the underlying principles and technical aspects of semen processing for elimination of HIV-1. (Reprod Med Biol 2007; 6: 151-156).