Low Sperm Numbers Research Articles

9325 Obesity Alters Pomc And Kisspeptin Neuron Crosstalk Leading To Lower Luteinizing Hormone

Abstract Disclosure: P. Villa: None. R. Ruggiero-Ruff: None. B. Jamieson: None. R. Campbell: None. D. Coss: None. Obesity is a significant public health concern due to its high prevalence and numerous comorbidities. It is associated with hypogonadism in males, characterized by low testosterone and sperm number. Previous studies determined that these stem from dysregulation of hypothalamic circuitry that regulates reproduction, by unknown mechanisms, since studies are confounded by chronic nature of this condition that leads to synaptic changes and alterations in neuron responsiveness to stimuli. Herein, we used mice fed chronic high-fat diet, that mimics human obesity, to determine mechanisms of impairment at the level of the hypothalamus, in particular gonadotropin-releasing hormone (GnRH) neurons that regulate luteinizing hormone levels (LH), which in turn regulates testosterone synthesis. Consistent with obese humans, we demonstrated lower LH, and lower pulse frequency of LH secretion, but unchanged pituitary responsiveness to GnRH. Pulse frequency of LH is regulated by pulsatile GnRH secretion, which is controlled by kisspeptin. Peripheral and central kisspeptin injections, and DREADD-mediated activation of kisspeptin neurons, demonstrated that kisspeptin neurons were suppressed in obese mice. Thus, we investigated regulators of kisspeptin secretion. We determined that LH response to NMDA was lower in obese mice, corresponding to fewer glutamate receptors in kisspeptin neurons, which may be critical for kisspeptin synchronization. Given that kisspeptin neurons also interact with POMC neurons, which regulate satiety and are particularly affected by obesity, we examined their crosstalk, and determined that LH response to either DREADD-mediated activation of POMC neurons or central injection of αMSH, a product of POMC, is abolished in obese mice. This was accompanied by diminished levels of αMSH receptor, MC4R, in kisspeptin neurons. The reason may be that chronically higher activity of POMC neurons in response to obesity, downregulates αMSH receptors on target neurons, including kisspeptin. This may lead to suppression of kisspeptin neurons, and their inability to regulate pulsatile secretion of GnRH. Together, our studies determined that obesity leads to downregulation of receptors that regulate kisspeptin neurons, which is associated with lower LH pulse frequency, leading to lower LH and hypogonadism. Presentation: 6/2/2024

P-347 In search of the most efficient device to freeze low numbers of spermatozoa: a comparative evaluation of various vitrification device

Abstract Study question Can different vitrification devices be used for cryopreservation of few spermatozoa for patients with varying sperm concentrations and which device is the most efficient? Summary answer The study demonstrates the efficacy of Cryotop, CBS, and Vitrifit devices in cryopreservation of low numbers of spermatozoa, with Cryotop Open showing superior performance. What is known already Sperm cryopreservation is crucial for managing male-factor infertility, yet conventional methods, reporting significant reduction in motility with only 30 to 40% viability post-thawing, fall short of optimal cryopreservation of small numbers of spermatozoa. Currently, ongoing efforts to enhance cryopreservation techniques seek to minimize cryodamage and optimize outcomes, particularly for spermatozoa from patients with conditions like oligozoospermia, cryptozoospermia, or azoospermia, where each spermatozoa is of substantial importance. Several devices have shown promising results regarding cryopreservation of small numbers of spermatozoa. However, to date, no comparison has been performed between multiple embryo-vitrification devices in terms of recovery and viability post thawing. Study design, size, duration Between January 2023 and May 2023, this study included 50 fresh ejaculated routine samples: 25 with spermatozoa concentrations >1 million/ml and 25 samples with concentrations <1 million/ml. For each sample, 10 motile spermatozoa were loaded on four different embryo-vitrification devices performed in triplicate: Cryotop Open (Kitazato), Cryotop Closed (Kitazato), CBS (Cryo BioSystem) and Vitrifit (Cooper Surgical). Recovery, motility, immotile vitality rates, user-friendliness as well as the duration of the freezing/thawing procedures were examined. Participants/materials, setting, methods Ten spermatozoa, selected in Hepes-HSA, were loaded into a 2 µl mixture of SpermFreeze and HEPES-HSA. Held 3 cm above LN2 for 2 minutes, straws were plunged into LN2. Thawing involved immersing straws in 2 µl Hepes-HSA under oil. Hypo-Osmotic-Swelling test was used for the vitality assessment of the immotile spermatozoa. Thawing protocols showed minor variations based on the carrier type. The study was approved by the local Ethics Committee. Main results and the role of chance This study conducted a detailed analysis of four vitrification devices for cryopreservation of low spermatozoa numbers. For concentrations >1 million/ml, Vitrifit, Cryotop Open, Cryotop Closed, and CBS displayed comparable recovery rates (mean % ± SD) (85±0.09, 89±0.06, 84±0.10, 86±0.07); motility rates (76±0.12, 79±0.08, 78±0.13, 80±0.08); immotile vitality rates (16±0.08, 13±0.07, 13±0.09, 12±0.07). In this group, Vitrifit and Cryotop Open exhibited similar freezing and recovery times (4’10’’/7’42’’ and 4’11’’/7’40’’ respectively). Cryotop Closed and CBS had slightly longer freezing times (4’33’’ and 5’31’’ respectively), with Cryotop Closed showing the fastest recovery (7’11’’). Similarly, for sperm concentrations <1 million/ml, devices demonstrated similar recovery (87±0.09, 91±0.06, 88±0.07, 83±0.12), motility (55±0.10, 61±0.09, 56±0.07, 50±0.09), and immotile vitality (26±0.07, 26±0.06, 27±0.07, 27±0.06). For concentrations <1 million/ml, all devices showed similar freezing and recovery times. Statistical analyses, including ANOVA and Levene’s test, revealed no significant inter-device differences, substantiated by minimal Cohen’s d effect sizes. Regarding the practical aspect, due to the ergonomic design and plastic composition that allows better microscopic visibility, Cryotop (both closed and open) demonstrated superior handling performance to CBS. Considering both statistical validation and manipulation, Cryotop Open distinguishes itself as the most efficient and user-friendly device for cryopreservation of low spermatozoa numbers. Limitations, reasons for caution The study’s main limitation is the limited sample size. Further research with larger cohorts and exploration of fertilization potential is necessary to validate these findings. Additionally, our study did not explore potential variations in cryopreservation outcomes related to additional sperm sample characteristics, which could impact the results. Wider implications of the findings Successful cryopreservation of low sperm numbers provides hope for cryptozoospermia and (non-)obstructive azoospermia patients. It allows the use of their own sperm, potentially avoiding donation. With superior frozen sperm survival, recovery rates, and a shorter search time, this method enhances fertility preservation accessibility, guiding patients in future IVF treatments effectively. Trial registration number EC-2022-272

Obesity Alters POMC and Kisspeptin Neuron Cross Talk Leading to Reduced Luteinizing Hormone in Male Mice.

Obesity is associated with hypogonadism in males, characterized by low testosterone and sperm number. Previous studies determined that these stem from dysregulation of hypothalamic circuitry that regulates reproduction, by unknown mechanisms. Herein, we used mice fed chronic high-fat diet, which mimics human obesity, to determine mechanisms of impairment at the level of the hypothalamus, in particular gonadotropin-releasing hormone (GnRH) neurons that regulate luteinizing hormone (LH), which then regulates testosterone. Consistent with obese humans, we demonstrated lower LH, and lower pulse frequency of LH secretion, but unchanged pituitary responsiveness to GnRH. LH pulse frequency is regulated by pulsatile GnRH secretion, which is controlled by kisspeptin. Peripheral and central kisspeptin injections, and DREADD-mediated activation of kisspeptin neurons, demonstrated that kisspeptin neurons were suppressed in obese mice. Thus, we investigated regulators of kisspeptin secretion. We determined that the LH response to NMDA was lower in obese mice, corresponding to fewer glutamate receptors in kisspeptin neurons, which may be critical for kisspeptin synchronization. Given that kisspeptin neurons also interact with anorexigenic POMC neurons, which are affected by obesity, we examined their cross talk, and determined that the LH response to either DREADD-mediated activation of POMC neurons or central injection of αMSH, a product of POMC, is abolished in obese mice. This was accompanied by diminished levels of αMSH receptor, MC4R, in kisspeptin neurons. Together, our studies determined that obesity leads to the downregulation of receptors that regulate kisspeptin neurons, which is associated with lower LH pulse frequency, leading to lower LH and hypogonadism.

Freezing Low Numbers of Sperm

Azoospermia, a condition defined by a complete lack of sperm in the ejaculate, presents a significant reproductive challenge for many infertile men worldwide. In recent years, Microsurgical Sperm Extraction (Micro-TESE) has emerged as an innovative surgical technique, allowing clinicians to retrieve viable sperm directly from the testicular tissues of patients diagnosed with this condition. A crucial aspect of optimizing the reproductive potential of the extracted sperm lies in the subsequent preservation techniques employed. This study delves deep into one such technique, sperm vitrification. As opposed to the traditional slow freezing methods, vitrification offers a rapid-cooling approach that effectively sidesteps the formation of potentially damaging ice crystals, thereby maintaining the structural and functional integrity of the sperm. The survival rates of sperm post-thawing, when subjected to vitrification, have been found to be commendably high, with figures often exceeding 60%. This is a promising statistic, particularly when dealing with minuscule sperm quantities as retrieved in Micro-TESE. However, the importance of sperm freezing transcends azoospermic scenarios. A broader and crucial indication for sperm freezing is fertility preservation, particularly poignant for cancer patients on the brink of undergoing treatments like chemotherapy or radiation, which could jeopardize their reproductive future. For these individuals, the opportunity to freeze their sperm offers a chance to father biological offspring post-recovery. By allowing multiple in vitro fertilization (IVF) attempts without necessitating recurrent surgical interventions, sperm cryopreservation, especially using vitrification, amplifies the chances of achieving biological parenthood. This is pivotal for both azoospermic individuals and cancer patients, safeguarding their dreams of family amidst medical adversities. In conclusion, the efficacy of vitrification, especially in the context of limited sperm counts, has the potential to reshape the landscape of male infertility treatments, offering a glimmer of hope for numerous affected individuals and their partners.

Tip-microVapour Fast Freezing: A novel easy method for cryopreserving severe oligozoospermic samples.

Sperm cryopreservation is an important procedure for oligozoospermic subjects at risk of azoospermia and after surgical recovery of spermatozoa in non-obstructive azoospermic men. Conventional procedures for sperm cryopreservation might be, however, not suitable for samples with a very low sperm number. In this pilot study, we investigated the recoveries of sperm motility and viability in severe oligozoospermic subjects (n=39) after cryopreservation with a tip-microVapour Fast Freezing, a procedure previously developed by our group for men with good semen quality. Sperm DNA fragmentation was also evaluated in a second group of oligozoospermic samples (n=16). We used a Vapour Fast Freezing procedure using 10μL tips as carrier, and Test Yolk Buffer as freezing medium (tip-microVapour Fast Freezing). In a subset of samples (n=22), we compared recovery of motility and viability as obtained with tip-microVapour Fast Freezing and with a Vapour Fast Freezing procedure using 500μL straws. Sperm DNA fragmentation was evaluated by the sperm chromatin dispersion test. We found a recovery rate (median [interquartile range]) of 0.29 (0.13-0.41) for progressive motility, 0.30 (0.21-0.52) for total motility and 0.48 (0.29-0.60) for viability. Interestingly, we observed that samples with the poorest motility were apparently less damaged by freezing/thawing. In a subset of samples (n=22), we directly compared values of viability, progressive motility and total motility by freezing/thawing with tip-microVapour Fast Freezing and Vapour Fast Freezing conducted with 500μL straws. We found much better values of all sperm parameters in samples after freezing/thawing with tip-microVapour Fast Freezing than with Vapour Fast Freezing in 500μL straws: that is, progressive motility: 7.00 (3.00-8.50)% versus 2.00 (0.00-4.25)%, p<0.001; total motility: 12.00 (8.00-16.25)% versus 6.50 (1.00-9.25)%, p<0.001; viability: 29.75 (23.75-45.25) versus 22.50 (13.75-28.13), p<0.001, respectively. In the second group of oligozoospermic samples, we found that tip-microVapour Fast Freezing produced lower levels of sperm DNA fragmentation than straws (33.00 [19.75-36.00]% vs. 36.00 [22.75-41.87]%, p<0.001). Tip-microVapour Fast Freezing appears to be a very promising method to cryopreserve semen samples from severe oligozoospermic patients.

ARRDC5 expression is conserved in mammalian testes and required for normal sperm morphogenesis

In sexual reproduction, sperm contribute half the genomic material required for creation of offspring yet core molecular mechanisms essential for their formation are undefined. Here, the α-arrestin molecule arrestin-domain containing 5 (ARRDC5) is identified as an essential regulator of mammalian spermatogenesis. Multispecies testicular tissue transcriptome profiling indicates that expression of Arrdc5 is testis enriched, if not specific, in mice, pigs, cattle, and humans. Knockout of Arrdc5 in mice leads to male specific sterility due to production of low numbers of sperm that are immotile and malformed. Spermiogenesis, the final phase of spermatogenesis when round spermatids transform to spermatozoa, is defective in testes of Arrdc5 deficient mice. Also, epididymal sperm in Arrdc5 knockouts are unable to capacitate and fertilize oocytes. These findings establish ARRDC5 as an essential regulator of mammalian spermatogenesis. Considering the role of arrestin molecules as modulators of cellular signaling and ubiquitination, ARRDC5 is a potential male contraceptive target.

Pharmacological perturbation of peroxisome-proliferator-activated receptor gamma alters motility and mitochondrial function of bovine sperm.

Sperm transit through the female reproductive relies upon maintenance of sperm motility. Peroxisome-proliferator-activated receptor gamma (PPARγ) is a ligand-activated nuclear transcription factor with roles in glucose metabolism and reproductive processes including placental function. PPARγ roles in the mammalian postejaculatory sperm function are incompletely defined. Determine expression, localization, and functions of PPARγ in postejaculatory bovine sperm. Frozen-thawed bovine sperm from three to four different bulls were pooled and subjected to immunofluorescence and western blot for detection and localization of PPARγ. Functions in sperm energetics were explored through the addition of pharmacological inhibition (GW; GW9662) and activation (Ros; Rosiglitazone) in the culture medium at 0 and 24h under non-capacitating conditions. Samples were analyzed for sperm kinematics (CASA) and mitochondrial membrane potential (MMP; JC-1 fluorophore). PPARγ was detected in bovine sperm and co-localized to the acrosome with re-localization to the equatorial region in acrosome-compromised sperm. The addition of Ros 50µM for 24 h maintained superior total and progressive motility of sperm compared to vehicle control (VC-DMSO 0.01%). The PPARγ antagonist GW 1µM was detrimental to both total and progressive motility. A challenge experiment (Ros + GW) partially rescued total and progressive motility phenotypes observed with GW incubation. GW-treated samples had a lower number of sperm with high MMP at 24 h compared to Ros or VC. The negative GW MMP phenotype was reversed with the addition of Ros + GW. Likewise, GW-treated samples had more sperm with low MMP compared to VC and Ros, and this phenotype was partially restored with Ros + GW. PPARγ is expressed in post-ejaculatory bovine sperm with regulatory roles in sperm motility and MMP. These findings implicate PPARγ as a novel regulator of postejaculatory mammalian sperm energetics through non-canonical signaling mechanisms.

Clinical and neonatal outcomes of individually vitrified human sperm with Cryotop and Cell Sleeper

Technique for preserving limited number of human spermatozoa is important for successful treatment of patients with azoospermia and cryptozoospermia. This study determined whether the non-biological devices (Cryotop and Cell Sleeper) efficiently vitrify small numbers of human spermatozoa. From December 2011 to December 2018, 10 males with very low sperm numbers managed with a single sperm vitrification method. Post-warmed sperm recovery was similar with both devices. Post-warmed sperm motility and fertilization after intracytoplasmic sperm injection were significantly higher in Cryotop group than in Cell Sleeper group (40.0% vs. 22.0%, P < 0.01 and 50.7% vs. 21.7%, P < 0.01, respectively). The pregnancy rate was 15.4% and 2 healthy babies were born in the Cryotop, while 14.3% and 1 baby in the Cell Sleeper, which did not differ between the groups. Both devices have clinical advantages in terms of easy use and safety, and would be performed more efficiently by using devices with different properties.

P-469 Micro-vapor fast freezing of human spermatozoa: development of a new method to use low number of cryopreserved spermatozoa

Abstract Study question Can Vapor Fast Freezing conducted in 10µl tips (microVFF/tips) efficiently preserve sperm viability and motility in banking samples with very low sperm concentration? Summary answer MicroVFF resulted able to efficiently preserve sperm viability and motility in patients with severe oligozoospermia or criptozoospermia What is known already Sperm cryopreservation is usually performed by conventional slow or fast freezing using carriers containing large semen volume, such as 500ul High Security Straws (500µl HSS). These carriers however are not suitable for patients with very low sperm concentration and many devices have been tested to freeze small volumes or even singularly isolated sperms (i.e.SpermVD®, Cryoloop, Cell Sleeper). Recently, our group showed that micro-VFF better preserve sperm viability and motility or DNA integrity with respect to a VFF conducted with 500µl HSS, in normozoospermic samples. Study design, size, duration The study was designed for 20 oligospermic or criptozoospermic subjects afferent to Semen Cryopreservation and Andrology Laboratory of Careggi Hospital for semen analysis, but during 24 months up to 34 patients were recruited for the trial. 13 samples were subsequently thawed and, due to the low sperm number, only motility and viability was compared between the two methods. Participants/materials, setting, methods 34 semen samples with low sperm concentration (0.06-7.7mil/ml) were cryopreserved with either microVFF/tips or the conventional method (VFF/ 500µl HSS). Thawing was conducted by keeping sample at room temparature and immediately observed by light microscopy at 20x and 40x. Total motility was scored as rapid/slow/non-progressive motility (a+b+c). Viability was evaluated by eosin test. For each method, the recovery of motility and viability was calculated as: post-thaw percentage/pre-thaw percentage Main results and the role of chance The microVFF method showed a better recovery of motility than the conventional method: 0.38 (±0.21)% vs 0.21 (±0.14)% (n = 13, p &amp;lt; 0.05). A similar statistically significant difference was observed for the mean recovery of viability: 0.43 (±0.20) vs 0.29 (±0.13) (n = 13, p &amp;lt; 0.5). for micro-freezing and conventional method, respectively. As frozen samples are used in the ART setting, microVFF/tips also shows the technical advantage to discharge directly the sample in fresh clean medium bridge in the ICSI plate, where swimming spermatozoa are washed from cryoprotectant compounds. Skipping conventional centrifugation/washing step after thawing highly protect from sperm motility/viability loss, as recently demonstrated by our group. Limitations, reasons for caution Results need to be confirmed in a larger number of patients. Although we previously showed that microVFF/tips also better preserve sperm DNA integrity than conventional method, in this study no other evaluations were possible due to the low number of spermatozoa/sample Wider implications of the findings Present results appear to be promising to enlarge the population who can be offered semen cryopreservation in our laboratory, including Klinefelter patients, severe oligo- and criptozoospermic menand patients undergoing testicular biopsy. Trial registration number not applicable

Developmental validation of an efficient differential separation method incorporating the i-sep® DL spin column with high sperm DNA recovery for the processing of sexual assault samples.

The differential separation method is key to recovering a DNA profile of the sperm donor from sexual assault samples. However, low numbers of spermatozoa from the perpetrator are often swamped by the victim's epithelial cells or lost during the separation process, with the separation process labor-intensive, time-consuming, and operator-dependent. The self-sealing filter of the i-sep® DL spin column allows direct lysis of the substrate throughout the differential separation process while preventing intact sperm cells from passing through, maximizing DNA recovery, and separation of non-sperm and sperm cells present. This study investigated the efficacy of a modified differential separation method, which incorporated the i-sep® DL spin column in comparison with the conventional pellet-based differential separation method. Using semen dilution series and mock post-coital samples, the sensitivity, reproducibility, repeatability, and efficiency of sperm DNA recovery of the pellet-based differential to the i-sep® method were evaluated side by side. The i-sep® differential method was more sensitive in capturing sperm fraction DNA, with informative semen donor alleles detected from high dilutions of semen inputs where the pellet method has been unsuccessful. The i-sep® differential method reduces manual handling, generating repeatable, and reproducible results between operators. Re-extraction of samples previously processed by the pellet or i-sep® differential method showed that the pellet method failed to recover 15-88% of sperm fraction DNA, while the i-sep® differential method was able to recover >99% in the initial extraction. The i-sep® method is robust for processing sexual assault samples, overcoming the challenges of sperm DNA losses encountered by pellet-based methods.

Applications and world-wide use of sexed semen in cattle

Successful sorting of sperm based on presence of the X- or Y-chromosome was first reported in the early 1980′s with the first live births reported in rabbits in 1988. Subsequent development of technological efficiencies resulted in commercialization of sex-sorted semen to cattle producers in 2003–2005. At product launch, low throughput dictated that reasonable prices to the producer could only be accomplished with extremely low sperm number dosages (2 × 106). Furthermore, conception rates were 70%–75% of those achieved by conventional unsorted product. Refinements in sorting equipment have enhanced the number of sperm that can be sorted from a semen sample and (or) aliquot of time, which translates into reduced production costs, while modifications to other aspects of sperm processing and freezing have facilitated maintenance of a conception potential more similar to that of conventional semen. More recently, strategic use of sex-sorted semen coupled with genomic technologies to identify superior females to satisfy replacement female needs has, by default, led to identification of a population of dairy cows from which replacements are not desired, leading to a tremendous increase in use of beef semen in dairy herds. Though exact numbers are unavailable, estimates indicate sex-sorted semen is rapidly approaching 30% of the total AI market share in North America. Though the primary application of sex-sorted semen is to accelerate genetic progress while enhancing biosecurity through in-house production of replacement animals, numerous other potential applications are evolving or are under consideration.

MP31-02 MICROTESE AND CRYOPRESERVED SPERM - A DECISION-MAKING DILEMMA FOR PATIENTS AND PROVIDERS

You have accessJournal of UrologyInfertility: Therapy (MP31)1 Sep 2021MP31-02 MICROTESE AND CRYOPRESERVED SPERM - A DECISION-MAKING DILEMMA FOR PATIENTS AND PROVIDERS Johnathan Doolittle, Darren Bryk, Scott Lundy, and Sarah Vij Johnathan DoolittleJohnathan Doolittle More articles by this author , Darren BrykDarren Bryk More articles by this author , Scott LundyScott Lundy More articles by this author , and Sarah VijSarah Vij More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002035.02AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: A known disadvantage of frozen microdissection testicular sperm extraction (mTESE) is the negative impact of cryopreservation and subsequent thawing of any sperm that are obtained. A post thaw (PT) sample provides insight in to how the sample withstands this stress and the predicts likelihood of sperm available for intracytoplasmic sperm injection (ICSI). If a PT cannot be performed due to such low numbers of sperm identified or no pre-freeze motility, providers and couples arrive at a challenging decision in their fertility pathway - pay for an additional sperm retrieval procedure or proceed with a sample that may not be sufficient for ICSI, requiring a back-up plan. Our study sought to characterize the ICSI outcomes after retrieval of motile sperm during frozen mTESE at our institution. METHODS: A retrospective chart review was conducted from 2012 through 2020 to identify all men that underwent frozen mTESE with successful retrieval of motile sperm. Prior sperm extraction or biopsy, testis size, hormone levels, genetics and prior semen analyses were recorded. A PT and recommendations regarding ISCI were made by the lead embryologist at our institution. When available, ISCI outcomes were recorded. RESULTS: Of the 88 men who underwent frozen mTESE, motile sperm were identified in 26.1% (23/88). 16 of these samples were assessed with a PT, of which 12/16 had PT motility. Of these 12, 10 specimens were deemed sufficient to proceed to ISCI by the embryologist whereas the remaining 2 couples were advised to consider fresh mTESE. ICSI records were available for 7 of the 12 patients with PT motility. All 7 cycles led to embryos for transfer, resulting in 6 pregnancies and 5 live births. Of the 11 men without PT motility, ICSI records were available for 6 couples. 3 underwent subsequent fresh mTESE all leading to embryos for transfer but no pregnancies, and 3 proceeded straight to ICSI, 2 of which created an embryo for transfer, with 1 resulting in an ongoing pregnancy. CONCLUSIONS: Of those men who have successful frozen mTESE, as defined as recovery of motile sperm, some may be advised to pursue fresh mTESE based on poor PT parameters or absence of PT due to poor pre-freeze parameters. This creates a very difficult decision for couples. Although frozen mTESE potentially spares the female partner an unnecessary IVF cycle, we often place couples in a difficult position to make a decision about whether to proceed to ICSI absent PT or in the setting of poor PT motility. Source of Funding: None © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e555-e555 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Johnathan Doolittle More articles by this author Darren Bryk More articles by this author Scott Lundy More articles by this author Sarah Vij More articles by this author Expand All Advertisement Loading ...

287 Effects of Number of Sperm and Site of Uterine Semen Deposition on Conception Rate and Number of Embryos in Weaned Sows Receiving a Single Fixed Time Insemination

Abstract Induced ovulation with single fixed time artificial insemination (SFTAI), combined with uterine (IUI) or deep uterine insemination (DUI), could improve fertility with low numbers of sperm and allow greater use of high genetic merit boars. At weaning (0 h), sows (n = 534) were assigned by parity and estrus induction method (eCG or Control) to receive 1200 × 106 sperm by IUI, 600, 300, or 150 × 106 sperm by IUI or DUI, or 75 × 106 sperm by DUI. At 80 h post weaning, sows received OvuGel and 26 h later a pooled semen SFTAI. Ultrasound was performed to determine follicle size and time of ovulation (OV). Sows were slaughtered 27 d after AI to determine pregnancy and litter traits. There was no effect (P &amp;gt; 0.05) of eCG on estrus (93%) within 5 d of weaning or follicle size (6.1 mm) at OvuGel, but wean-to-estrus (3.8 vs. 4.0 d,P &amp;lt; 0.01) and AI-to-OV (15.9 vs. 17.0 h, P = 0.04) intervals and AI-to OV were slightly reduced. eCG did not affect pregnancy rate (78.6%), number of CL (21.7), or number of viable embryos (12.2). There was no effect of number of sperm or site of insemination (P &amp;gt; 0.05) on pregnancy rate (range: 80.9% to 70.5%), but AI occurring after ovulation reduced pregnancy rate (P &amp;lt; 0.02). Total number of embryos (range: 16.5 to 10.3) increased with CL number (P &amp;lt; 0.001) but was not affected by number of sperm or site of insemination (P &amp;gt; 0.05). Higher sperm treatments (1200 and 600 x 106) had more embryos compared to lower sperm treatments (P &amp;lt; 0.01), suggesting that lower sperm numbers effects litter size more than the pregnancy status. Acceptable fertility can be achieved with low sperm numbers when using SFTAI and uterine deposition, but AI-to-OV interval and ovulation rate influence final fertility.

First emerging evidence of the relationship between Onuf's nucleus degeneration and reduced sperm number following spinal subarachnoid haemorrhage: Experimental study.

Lumbosacral pathologies can lead to infertility. Onuf's nucleus changes in these pathologies may have a role in low sperm number. This study aims to investigate the relationship between Onuf's nucleus degeneration and sperm number following spinal subarachnoid haemorrhage. 22 rabbits were used. They were divided into three groups; five of them were used as the control (GI), five as the SHAM (GII) and twelve as the study groups (GIII). The study group received 0.7ccs autologous blood into the spinal subarachnoid space at the T12-L1 level. After two weeks, all animals were decapitated, and S1-S3 laminectomy was done. Neurodegenerative changes of Onuf's nucleus, pudendal ganglia (S3) following two weeks after spinal SAH, were examined; sperm numbers were calculated. Degenerated neuron density of the Onuf's nucleus (n/mm3 ), the pudendal ganglia (S3) (n/mm3 ) and mean sperm numbers were calculated as 5±2, 8±3/mm3 and 98.345±12.776/mm3 in the control (GI), 20±5/mm3 , 243±66/mm3 and 91.841±9.654/mm3 in the SHAM (GII), 143±39/mm3 , 2,350±320/mm3 and 68.549±5.540/mm3 in the study group (GIII). In conclusion, there were statistically significant differences between groups. Onuf's nucleus may be responsible for decreased sperm number following spinal SAH.

The Effectiveness of LenshookeTM Semen Quality Analyzer X1 Pro for Human Semen Analysis

Background: LenshookeTM Semen Quality Analyzer (SQA) X1 Pro is an automated semen analysis. The accuracy of LenshookeTM SQA X1 Pro has never been analyzed with World Health Organization (WHO) standard method. Aim: This study aims to examine whether the LenshookeTM SQA X1 Pro method provides reliable results according to the WHO standard method. Methods: This study was a laboratory analytic observational study using 60 patients in Andrology clinic of Dr. Soetomo Hospital. The concentration, progressive motility (PR), total motile sperm count (TMSC), and morphology results of the LenshookeTM SQA X1 Pro and standard method were analyzed statistically using correlation, Bland Altman, and diagnostic test. Results: Significant correlation between two methods were found in all parameters (concentration: r = 0,970; PR: r = 0,781; TMSC: r = 0,952; morphology: r = 0,568). The mean difference for concentration, PR, TMSC, and morphology between the two examination methods were 1,165 million/ml, 7,05%, 7,584 million/ejaculate, and 2,25%. However, it found that the correlation and agreement were weaker in sample with low number of spermatozoa per high power field. The results revealed a sensitivity of 100%, 81%, and 59% for oligozoospermia, astenozoospermia, and teratozoospermia, respectively. The specificities were shown to be 100%, 74%, and 100% for oligozoospermia, astenozoospermia, and teratozoospermia, respectively. Conclusion: The LenshookeTM SQA X1 Pro gives a reliable result for determining oligozoospermia and asthenozoospermia, but in the situation that the clinicians need the accurate data, standard method should be used.

Reproductive performance in cervical and postcervical artificial insemination (PCAI) with liquid boar semen in Gunghroo × Hampshire crossbreed pig in Nagaland

Genetic advancement for the modern swine industry is primarily accomplished through the use of reproductive biotechnology, mainly artificial insemination (AI). The objective of the present study was to compare the fertility outcome by cervical and post-cervical artificial insemination (PCAI) using normal (three billion) and reduced (one billion) number of spermatozoa in pig. Pluriparous weaned sows were grouped into 4 groups, i.e. Group- 1, AI with three billion spermatozoa by intra-cervical insemination; Group-2, AI with one and half billion spermatozoa by intra-cervical insemination; Group-3, AI with three billion spermatozoa by PCAI and Group-4, AI with one and half billion spermatozoa by PCAI. Non-significantly higher farrowing rate was recorded in Group-3 compared to Group-1. Post-cervical AI with lower number of spermatozoa (Group-4) resulted into farrowing rate which was similar to cervical insemination with higher number of spermatozoa (Group-1). There was significant difference in litter size at birth and litter size at weaning between Group-1 and 2. Litter size at birth and litter size at weaning was significantly higher in Group-4 compared to Group-2. Also, litter size at birth and litter size at weaning was significantly higher in PCAI animals (Group-2 and 4) compared to cervical inseminated animals (Group-1 and 2). In conclusion, PCAI with liquid boar semen was found to have improved farrowing rate, litter size at birth and litter size at weaning compared to cervical insemination.

The Tug1 lncRNA locus is essential for male fertility

BackgroundSeveral long noncoding RNAs (lncRNAs) have been shown to function as components of molecular machines that play fundamental roles in biology. While the number of annotated lncRNAs in mammalian genomes has greatly expanded, studying lncRNA function has been a challenge due to their diverse biological roles and because lncRNA loci can contain multiple molecular modes that may exert function.ResultsWe previously generated and characterized a cohort of 20 lncRNA loci knockout mice. Here, we extend this initial study and provide a more detailed analysis of the highly conserved lncRNA locus, taurine-upregulated gene 1 (Tug1). We report that Tug1-knockout male mice are sterile with underlying defects including a low number of sperm and abnormal sperm morphology. Because lncRNA loci can contain multiple modes of action, we wanted to determine which, if any, potential elements contained in the Tug1 genomic region have any activity. Using engineered mouse models and cell-based assays, we provide evidence that the Tug1 locus harbors two distinct noncoding regulatory activities, as a cis-DNA repressor that regulates neighboring genes and as a lncRNA that can regulate genes by a trans-based function. We also show that Tug1 contains an evolutionary conserved open reading frame that when overexpressed produces a stable protein which impacts mitochondrial membrane potential, suggesting a potential third coding function.ConclusionsOur results reveal an essential role for the Tug1 locus in male fertility and uncover evidence for distinct molecular modes in the Tug1 locus, thus highlighting the complexity present at lncRNA loci.

Effects of the number of sperm and site of uterine semen deposition on conception rate and the number of embryos in weaned sows receiving a single fixed-time insemination.

Reducing the number of sperm needed to produce a litter with artificial insemination (AI) allows greater use of higher genetic merit boars. Induced ovulation with single fixed-time artificial insemination (SFTAI), combined with intrauterine (IUI) or deep uterine insemination (DUI), could improve fertility with low numbers of sperm. The objectives of the study were to determine the fertility effects of sperm numbers and the site of insemination. At weaning (0 h), sows (n = 534) were assigned by parity and estrus induction method (equine chorionic gonadotropin [eCG] or Control) to receive 1,200 × 106 sperm by IUI; 600, 300, or 150 × 106 sperm by IUI or DUI; or 75 × 106 sperm by DUI. At 80 h postweaning, sows received OvuGel and 26 h later a SFTAI using pooled semen. Sows were exposed to boars once daily and ultrasound was performed to determine follicle size and time of ovulation. Following SFTAI, sows were slaughtered 27 d after AI to determine pregnancy and litter traits. Data were analyzed using different models to test for effects of estrus induction, interaction of three levels of sperm (600 to 150) with two levels for site (IUI vs. DUI), and the overall effects of AI method (eight treatments). There was no effect (P > 0.05) of estrus induction on estrus (93%) within 5 d of weaning or on follicle size (6.1 mm) at OvuGel, but wean-to-estrus interval (3.8 vs. 4.0 d) was slightly reduced (P < 0.01) as was AI-to-ovulation interval (15.9 vs. 17.0 h, P = 0.04) for eCG and Control, respectively. There was no effect (P > 0.05) of estrus induction on pregnancy rate (78.6%), number of corpora lutea (CL; 21.7), or number of viable embryos (12.2). There was no effect of number of sperm or site of insemination and no interaction (P > 0.05) on pregnancy rate (range: 80.9% to 70.5%), but AI occurring after ovulation reduced the pregnancy rate (P < 0.02). The total number of embryos (range: 16.5 to 10.3) was not affected by estrus induction, number of sperm, or site of insemination (P > 0.05), but was influenced by AI treatment (P < 0.01). Treatments with a higher number of sperm (1,200 and 600) had more embryos compared with those with a lower number of sperm (300 to 75). The numbers of embryos also increased with the number of CL (P < 0.0001). These results suggest that the lower number of sperm affects litter size more than the pregnancy status. Acceptable fertility can be achieved with low numbers of sperm when using a SFTAI and uterine deposition, but AI-to-ovulation interval and ovulation rate influence final fecundity.

Meiotic epigenetic factor PRDM9 impacts sperm quality of hybrid mice.

Reduced fertility of male mouse hybrids relative to their parents, or hybrid sterility, is governed by the hybrid sterility 1 (Hst1) locus. Rescue experiments with transgenes carrying sequences within or near Hst1 manifested that Hst1 contains the gene encoding meiosis-specific histone methyltransferase PRDM9. The Prdm9 gene is responsible for partial meiotic arrest, testicular atrophy, and low sperm count in (C57BL/6J x PWD)F1 mouse hybrids. Here we report that these male hybrids suffer an additional reproductive disadvantage, decreased sperm quality, which is (i) further exacerbated by the introduction of long transgenes carrying sequences from Hst1 with incomplete Prdm9 into their genome and (ii) controlled by the Prdm9 dosage. These transgenic male hybrids displayed the features of severe oligoasthenoteratozoospermia (OAT), a human infertility syndrome characterized by a low number of spermatozoa with poor motility and morphological abnormalities. Analysis of spermiogenesis in these mice revealed acrosome detachment, aberrant elongation and condensation of the nucleus. As a result, the transgenic sperm had acrosome malformations, abnormal chromatin packaging, and fragmented DNA with elevated base oxidation, revealed by using multiple methods. Heterozygosity for one null Prdm9 allele improved meiotic progression and sperm quality of both non- and transgenic hybrids. Our results indicate that genomic analysis of OAT patients should include consideration of allelic variants in PRDM9, and our transgenic models can serve as tools to understand the diverse molecular processes that, when perturbed, can cause this disease.

The effect of cryopreservation technique in a very low number sperm count at Soetomo Hospital, Surabaya, Indonesia

Background: The massive development of Assisted Reproductive Technology (ART) offers new hope for the infertile couple. However, the advanced storage methods to preserve the quality of sperm has been raising by cryopreservation technique. This study aims to analyze the motility rate, recovery rate, and viability in subgroups of cryopreserved spermatozoa from the oligozoospermic patient as the quality parameter.Method: A true-experimental study by pre-test and post-test group design was conducted among 13 oligozoospermic patients with a very low sperm counts at Medical Biology Laboratory Faculty of Medicine, Universitas Airlangga, Surabaya, Indonesia. Parameters assessed in this study include motility, viability, and concentration of spermatozoa according to Laboratory Manual for the Examination and Processing of Human Sperm 5th edition. The samples were divided into 4 groups such as Group 1 (71-100 sperm), Group 2 (31-70 sperm), Group 3 (11-30 sperm), and Group 4 (1-10 sperm) based on sperm counts which underwent 30 repetitions of cryopreservation. Data were analyzed using SPSS version 17 for Windows. Results: There was a significant difference in the recovery rate among Group 1 (34.88±1.87), Group 2 (44.77±1.89), Group 3 (61.83±2.09), and Group 4 (70.90±2.71) of cryopreservation technique (P&lt;0.001). However, there was no significantly different in the motility rate of sperm-cryopreservation technique among Group 1 (65.60±14.63), Group 2 (52.50±26.28), Group 3 (39.90±9.95), and Group 4 (44.70±41.77) (p=0.070). Based on the sperm-viability parameter assessed in this study, there was no significant difference among Group 1 (56.40±3.89), Group 2 (58.60±5.99), Group 3 (59.60±9.96), and Group 4 (56.70±11.30) (p=0.504).Conclusions: The sperm-cryopreservation technique significantly affected the recovery rate of very low sperm number count among groups. However, there was no significantly different in the motility rate and viability parameter in this study.Â