OBJECTIVE: Is acrosin activity related to cumulative live birth rate (CLBR) over 1 year after IVF, intracytoplasmic sperm injection (ICSI) treatment or both?
METHODS: Retrospective monocentric cohort study of 5704 couples who started IVF/ICSI treatments between 2016 and 2021. Acrosin activity was determined by a modified Kennedy method using a commercial kit. Patients were divided into two groups according to their acrosin activity: below 25 μIU/106 spermatozoa; and an acrosin activity 25 μIU/106 spermatozoa or above. Primary outcome was the CLBR, defined as an ongoing pregnancy leading to live birth that had arisen from all embryo transfers carried out within 1 year after the first ovum retrieval. Both conservative and optimistic methods were used for estimating CLBRs.
RESULTS: The CLBRs of patients with an acrosin activity below 25 μIU/106 spermatozoa were found to be significantly lower than those of patients with an acrosin activity 25 μIU/106 spermatozoa or above by conservative (48.5% versus 55.4%, P = 0.02) and optimistic (63.7% versus 70.3%, P = 0.047) methods after adjusting for confounders. When acrosin activity was regarded as a continuous variable, significant negative relationships between acrosin activity and CLBR were identified in subgroups: young couples (men and women aged younger than 30 years) and couples from whom no more than 10 eggs were retrieved.
CONCLUSIONS: Low acrosin activity levels were correlated with decreasing CLBRs over 1 year. These findings suggest that acrosin activity can be used as a predictor for CLBRs before starting IVF/ICSI treatment to enhance the effectiveness of counselling.

OBJECTIVE: Since the unexplained in vitro fertilization failure occurs frequently, it is of great importance and clinical value to identify potential underlying predictors. This study aimed to explore whether the percentage of sperm with a small acrosome was correlated with unexplained in vitro fertilization failure.
METHODS: A new acrosomal function evaluation index (the percentage of sperm with a small acrosome) was introduced into the analysis of sperm morphology. The association between the index and acrosome function by acrosin activity detection test and acrosome reaction test was investigated. In addition, the correlation with unexplained in vitro fertilization failure was further explored. Finally, the ROC curve was used to analyze the diagnostic efficacy on the failure of in vitro fertilization and the cutoff value was calculated.
RESULTS: As the increasing of the percentage of sperm with a small acrosome, the value of acrosin activity, acrosome reaction rate, and in vitro fertilization rate were reduced, with a statistically significant difference (P < 0.05). The index in the low fertilization rate group was significantly higher than that in the normal fertilization rate group (P < 0.05). Finally, the results of ROC curve found that when the index was 43.5%, the sensitivity and specificity were 74.2% and 95.3%, respectively.
CONCLUSIONS: The percentage of sperm with a small acrosome was positively correlated with unexplained in vitro fertilization failure, which could be potentially used as a prognostic index for the failure of in vitro fertilization.
BACKGROUND: [Ethics review acceptance No IIT20210339B].

Low acrosin activity (LAA) is associated with sperm function anomaly and poor outcomes of in vitro fertilization. In this study, we confirm that 993 semen samples with LAA had a reduced sperm motility and low in vitro fertilization rate in comparison with 1332 normal controls (NC). Proteomic comparison between 11 LAA and 11 NC sperm samples identified 35 upregulated and 99 downregulated proteins in the LAA group. Indeed, proteomic data showed that acrosome enzymes Spam1 and Acrosin were among the downregulated proteins in the LAA group, which was validated by quantitative PCR and immunefluorescent staining of sperm cells. The KEEG pathway analysis revealed a deficiency of GSH and Gln biosynthesis in LAA sperm cells. Immunofluorescent staining of sperms and quantitative PCR verified downregulation of GLUL and GCLC, the key enzymes for GSH and Gln biosynthesis. Moreover, the results of ELISA assay confirmed low levels of GSH and Gln in LAA sperm cells. Mechanistic studies showed that addition of 10 mM H2O2 to semen samples led to a significant reduction of acrosin activity and sperm motility, most possibly by triggering premature acrosome release. In contrast, the presence of 20 mM GSH blocked the oxidative effects of H2O2. Since GSH counteracts the oxidative stress and Gln participates in TCA cycling, their deficiency may affect the redox balance as well as energy production of sperm cells. These findings shed new light on the pathological mechanisms of infertility associated with LAA. Male infertility patients could benefit from GSH supplement by improvement of acrosin activity and other sperm functions.

The aim of the study was to evaluate semen quality in the two most popular colour morphs of the Arctic fox Alopex lagopus L., blue and white, based on ejaculate parameters, acrosin activity and analysis of sperm morphology. The research material consisted of ejaculates collected once by manual stimulation from 20 one-year-old male Arctic foxes (10 individuals of the blue morph and 10 of the white morph). Ejaculates were evaluated in terms of volume, sperm concentration, total number of spermatozoa and the percentage of spermatozoa with major and minor defects. The study revealed that male blue Arctic foxes produce ejaculates with much higher concentration (148.75 × 106 /ml) and total number of spermatozoa (98.16 × 106 ) compared to white Arctic foxes (42.88 × 106 /ml and 35.2 × 106 respectively). The level of acrosin activity from white foxes seemed to be higher compared to blue foxes but the difference was not statistically confirmed. Semen from Arctic foxes is characterized by high inter-individual variability in sperm morphology. The frequency of morphological changes in sperm from Arctic foxes does not significantly depend on ejaculate volume, sperm concentration or the total number of spermatozoa in the ejaculate, but is associated with acrosin activity.

The aim of this study was to observe sperm aneuploidy, DNA integrity, seminal alpha-glucosidase (NAG) and acrosin activity (AA) under testicular heat stress (SH). Spermatozoa were obtained from 30 healthy adult volunteers subjected to scrotal warming at 43°C for 30-40 min on two successive days per week for 3 months between February 2012 and September 2016. Aniline blue (AB), acridine orange (AO) staining, TUNEL assay and FISH analysis to evaluate sperm function, sperm DNA integrity and chromosomal abnormalities were carried on before, during and after SH. Sperm AA and NAG was measured by microplate reader. The mean parameters of sperm parameters, AA and NAG were significantly decreased. In contrast, the mean percentage of sperm DNA fragmentation and the proportion of aneuploidy of chromosomes 13, 18, 21, X and Y were significantly increased for spermatozoa collected during SH versus before SH (p < .01-.001). After stopping scrotal heating for 3 months, most parameters were completely restored to pre-SH levels. Sperm parameters, sperm DNA integrity, chromosomes, AA and NAG are affected by scrotal exposure to constant SH temperatures several degrees over normal physiological temperature, and after treatment, these parameters were reversibly restored to the level before SH in adult men.

Lead (Pb) is a heavy metal that can damage animal sperm. To study the effects of Pb on calcium homeostasis and calcium channel in the sperm of freshwater crab Sinopotamon henanense, the induction of acrosome reaction (AR) and acrosin activity were investigated when crabs were exposed to different Pb concentrations (0, 3.675, 7.35, 14.7, 29.4 and 58.8mg/L) for 3, 5 and 7 d separately. Fluorescent probe Fluo-3/AM was loaded into the sperm, and [Ca2+] in the sperm was measured by fluorescence microscopy and using microplate reader. The calmodulin (CaM) concentration was measured by ELISA method. Verapamil (VRP), a calcium channel blocker, was used to evaluate whether Pb can enter the sperm through calcium channels leading to sperm damage. After sperm were exposed at 50μg/L VRP, 100μg/L Pb, 50μg/L VRP+100μg/L Pb, 1000μg/L Pb and 50μg/L VRP+1000μg/L Pb for 1h in vitro，sperm quality parameters (sperm survival and sperm DNA integrity) and levels of parameters indicating oxidative stress (protein carbonylation [PCO] and malondialdehyde [MDA]) were measured. Our data showed that Pb reduced the induction of acrosome reaction (AR), down-regulated the acrosin activity, decreased the intracellular concentration of Ca2+ and elevated CaM concentration. Compared to controls, Pb alone induced significant stress, as reflected by decreasing sperm survival and sperm DNA integrity, and increasing PCO and MDA contents. In the presence of VRP, 100μg/L Pb-induced stresses were reduced, all the measured parameters in the sperm exposed at 100μg/L Pb returned to control levels. Our results indicate that Pb enters the sperm of the crab S. henanense through calcium channels, the inhibition of which blocks Pb-induced stresses such as sperm quality decline and oxidative damage.

Phthalates are suspected endocrine disrupting chemicals that impair male reproductive function in animal and epidemiological studies. We investigated associations between urinary phthalate metabolites and acrosin activity, along with that between insulin like-factor 3 (INSL3), a Leydig cell function marker, in Chinese adult men and assessed the association between the metabolites and male reproductive function. Serum levels of INSL3 and other hormones, semen parameters, and urinary concentrations of 14 phthalate metabolites in 1066 men were measured. The unadjusted concentrations of phthalates were included as independent variables and urinary creatinine as a separate covariate. INSL3 was negatively associated with mono-2-ethylhexyl phthalate (MEHP) and %MEHP [percentage of MEHP to all di(2-ethylhexyl) phthalate (DEHP) metabolites]. Acrosin activity was negatively associated with mono-n-butyl phthalate (MBP), mono-isobutyl phthalate (MiBP), MEHP and %MEHP. MBP and MiBP were also negatively associated with total testosterone (T), free androgen index (FAI), free testosterone (FT), luteinizing hormone (LH) and sperm morphology and positively associated with DNA fragmentation index (DFI). A negative association between %MEHP and sperm motility was observed. Several other metabolites were also associated with reproductive function. This is the first report on the inverse associations of phthalate metabolites with acrosin activity and INSL3. Phthalates may cause multiple adverse results on reproductive function at environmental levels.

Our previous study found that CLOCK knockdown in the testes of male mice led to a reduced fertility, which might be associated with the lower acrosin activity. In this present study, we examined the differential expression in proteins of CLOCK knockdown sperm. Clock gene expression was knocked down in cells to confirm those differentially expressions and serine protease inhibitor SERPINA3K was identified as a potential target. The up-regulated SERPINA3K revealed an inverse relationship with Clock knockdown. Direct treatment of normal sperm with recombinant SERPINA3K protein inhibited the acrosin activity and reduced in vitro fertilization rate. The luciferase reporter gene assay showed that the down-regulated of Clock gene could activate the Serpina3k promoter, but this activation was not affected by the mutation of E-box core sequence. Co-IP demonstrated a natural interaction between SERPIAN3K and RORs (α and β). Taken together, these results demonstrated that SERPINA3K is involved in the Clock gene-mediated male fertility by regulating acrosin activity and provide the first evidence that SERPINA3K could be regulated by Clock gene via retinoic acid-related orphan receptor response elements.

The main aim of this study was to determine whether acrosin activity could predict boar sperm freezability. For this purpose, we characterized the changes in sperm quality and acrosin activity throughout the cryopreservation procedure of sperm samples from 30 Pietrain boars by analyzing four critical steps: step 1 (extended sperm at 15 °C), step 2 (cooled sperm at 5 °C), step 3 (30 minutes postthaw), and step 4 (240 minutes postthaw). Freezability ejaculate groups were set on the basis of sperm motility and membrane integrity after freeze-thawing. Results obtained highlighted the low predictive value in terms of freezability of sperm motility and kinematics and sperm membrane integrity, as no differences between good and poor freezability ejaculates were seen before cryopreservation. Significant differences (P < 0.05) between ejaculate groups were observed in the cooling step at 5 °C for sperm kinetic parameters, and after thawing for sperm motility and membrane integrity. In contrast, acrosin activity appeared as an indicator of boar sperm freezability because the differences (P < 0.05) between good and poor freezability ejaculates manifested yet in extended samples at 15 °C. On the other hand, we also found that variations in sperm kinematics, membrane lipid disorder, intracellular calcium content, acrosome integrity, and acrosin activity throughout the cryopreservation procedure were indicative of a significant damage in spermatozoa during the cooling step in both ejaculate groups. In conclusion, the main finding of our study is that acrosin activity can be used as a reliable predictor of boar sperm freezability because it differs significantly between good and poor freezability ejaculates yet before freeze-thawing procedures took place, i.e., in the refrigeration step at 15 °C.

Soybean lecithin may represent a suitable alternative to egg yolk for semen cryopreservation in livestock species. However, additional studies are needed to elucidate its effects on spermatozoa functional properties. Semen collected from five Sarda bucks was cryopreserved in Tris-based extender and glycerol (4% v:v) with different supplementations. In a preliminary experiment, different soybean lecithin concentrations were tested (1%-6% wt/vol) and results in terms of viability, percentages of progressive motile and rapid spermatozoa, and DNA integrity after thawing showed that the most effective concentration was 1%. In the second experiment, semen was frozen in a Tris-based extender with no supplementation (EXT), with 1% lecithin (EXT LC), and 20% egg yolk (EXT EY). The effectiveness of these extenders was also compared with a commercial extender. The EXT EY led to the highest viability and motility parameters after freezing and thawing (P < 0.0001). No significant differences were observed in intracellular ATP concentrations. Additional molecular features revealed that sperm functionality was affected in EXT EY, as demonstrated by lower DNA and acrosome integrity (P < 0.05), and higher lipid peroxidation compared with spermatozoa cryopreserved in EXT LC (P < 0.0001). Results obtained in the heterologous in vitro fertilization test showed that EXT LC better preserved spermatozoa functionality, as demonstrated by the higher fertilization rates compared with the other media (66.2 ± 4.5% for EXT LC vs. 32.7 ± 4.5%, 38.7 ± 4.5%, 39.6 ± 5.2% for EXT, EXT EY, and commercial extender; P < 0.01). The present study demonstrated that lecithin can be considered as a suitable alternative to egg yolk in goat semen cryopreservation, because it ensures higher fertilization rates and a better protection from membrane damage by cold shock.