{"paper_id":"839d95d7-2890-4176-b667-7b44f63d161b","body_text":"The term \"infertility\" refers to the condition in which\na couple is unable to achieve conception even after\nconsistently having unprotected sexual intercourse for\none year ( 1 ). Estimates by the World Health Organization\nand epidemiological studies show that the worldwide\nprevalence of infertility is about 17.5% ( 2 ). Developing\ncountries, in particular, bear a substantial burden, with\none in every six couples grappling with infertility ( 3 ).\nRemarkably, half of the instances of infertility in couples can be attributed to male factors ( 4 ). These factors\nspan a spectrum, ranging from genetic conditions like\nKlinefelter’s syndrome ( 5 ) to environmental influences\nsuch as exposure to contaminants like mercury, arsenic,\nand lead ( 6 ), all of which can significantly impact male\nfertility. Infections such as human immunodeficiency virus\n(HIV), human papillomavirus (HPV), cytomegalovirus\n(CMV), adenoviruses, parvovirus, and mumps ( 7 ),\ncoupled with lifestyle choices and other environmental\nfactors, contribute to the multifaceted landscape of male infertility. Male infertility typically refers to a condition\nin which the inability to conceive is associated with a\nspecific alteration found in the male partner. Possible\nconsequences of this change include a sperm concentration\nbelow the lower reference (<15 million sperm/mL) in\nejaculate (oligozoospermia), reduced or absent sperm\nmotility (<32%) in fresh ejaculate (asthenozoospermia),\nand abnormal sperm morphology (teratozoospermia).\nHowever, a combination of these factors is generally\nregarded as oligoasthenoteratozoospermia (OAT) ( 8 ).\nRegrettably, the majority of instances of severe OAT are\nattributed to an unexplained testicular abnormality or\ndisorder ( 9 ). As a result, rational therapeutic approaches\nhave not been pursued. Instead, infertile men have been\nprescribed numerous uncontrolled treatments without\nsufficient pathophysiological justification or solely based\non empirical evidence.\nBased on innovative medical approaches, the use of\nplatelet-rich plasma (PRP), which is a high concentration\nof autologous platelets suspended in a small amount of\nplasma after centrifugation, appears to be a safe and highpotential therapeutic option ( 10 - 12 ). Due to its abundant\ngrowth factor content, PRP has already demonstrated\nits advantages in the field of regenerative therapy ( 13 ).\nCertain advantageous effects of PRP, such as accelerated\nangiogenesis, inflammation control, cell migration,\ndifferentiation, and proliferation, have been highlighted\nin several earlier research studies ( 14 - 16 ). Currently,\nPRP is increasingly utilized in the field of reproductive\nmedicine, owing to its regenerative potential. Extensive\nresearch has explored the potential benefits of PRP in\nfemale reproductive medicine, with studies focusing on\nintrauterine PRP injections for patients with recurrent\nimplantation failure (RIF) ( 17 ) and intraovarian PRP\napplications for women with poor ovarian response\n(POR) ( 18 ). In contrast, the investigation of PRP's effects\non male infertility is still in its nascent stage. Some studies\nhave examined its potential in this context, including its\nimpact on the fertility of individuals with non-obstructed\nazoospermia ( 19 ). However, it is crucial to acknowledge\nthat the existing scientific evidence is not yet sufficient to\nestablish definitive conclusions.\nTo the best of our knowledge, there is a scarcity of studies\nexploring the impact of PRP on male infertility, especially\nin the context of a focused investigation on severe OAT.\nThis study aims to fill this research gap by investigating\nthe specific impact of PRP on male infertility, with a focus\non improving sperm parameters in individuals with severe\nOAT. Through these efforts, our study seeks to contribute\nto the advancement of regenerative therapies in the field\nof reproductive health.\n\nThe present study is a randomized clinical trial\nconducted at the Infertility Center of Fatemieh Hospital\nin Hamedan from January 2022 to August 2023. Before\nparticipating in the study, each participant provided\ninformed consent, expressing their willingness to take\npart in the clinical trial. Comprehensive information\nabout the research, including its purpose, procedures, and\npotential risks and benefits, was conveyed through verbal\nexplanations and written documents. Emphasis was\nplaced on voluntary participation, ensuring participants’\nright to withdraw at any stage without consequences.\nOpportunities for questions and clarification were\nprovided prior to obtaining formal consent through signed\nforms. The trial was documented in the Iran Registry of\nClinical Trials (IRCT20220317054318N2). The Ethical\nCommittee of Hamedan University of Medical Sciences\ngranted approval for the implementation of the study (IR.\nUMSHA.REC. 1401.946).\nEighty-eight infertile males, who had been referred to the infertility center of\nFatemieh Hospital for infertility treatment, were enrolled in this clinical trial\naccording to the sample size calculation formula. Participants were recruited from those\nreferred to the infertility center for infertility treatment, aiming to enhance the\nstudy’s relevance to a broader population seeking infertility care. The inclusion criteria\nwere having an age range of 20- 45 years old with severe OAT (the sperm count\n≤4×10 6  /ml, progressive motility sperm ≤30%, morphologically normal sperm\n≤1%), not receiving other treatments such as hormone therapy, not having underlying\ndiseases (such as diabetes, kidney, liver diseases, addiction to drugs and alcohol),\ncancer, and receiving chemotherapy treatments.\nIn the present study, 88 infertile male patients were\ndivided into two groups: control (n=44) and intervention\n(n=44). Semen samples were analyzed before PRP\ninjection in both groups. In the intervention group, a\nurologist injected PRP into the testicular tissue using\nlocal anesthesia, with the amount of PRP varied from 1\nto 2 cc depending on the size of the testicle. After three\nmonths, another semen sample was taken from the same\nindividuals for sperm analysis and DNA fragment index\nevaluation. There was no intervention in the control group.\nThe sperm sample is collected after abstaining from\nsexual activity for three days. The diagnosis of OAT was\nverified by conducting two spermiogram tests one month\nafter the initial examination, and if there is a difference\nof more than 20% between the samples, a third test is\nperformed.\nIn this study, the semen sample was incubated for 30\nminutes at a temperature of 37°C to transform it from a\ncoagulated form to a liquefied state.\nThe PRP was prepared using their autologous blood\nsamples following established standard techniques ( 20 ).\nBriefly, 5 cc of whole blood was collected in tubes\ncontaining anticoagulant (EDTA) and gently mixed to\nprevent clotting. The blood was then centrifuged at 3000 rpm for 5 minutes, separating into distinct layers. Red\nblood cells settled at the bottom, a middle layer known as\nthe buffy coat containing white blood cells and platelets,\nand plasma at the top.\nUsing a sterile pipette or syringe, the upper layer\n(plasma) was carefully transferred to a new, sterile tube.\nThis plasma was then centrifuged at 3500 rpm for 15\nminutes. The upper two-thirds, consisting of PPP, were\nremoved, leaving the lower one-third containing PRP.\nTo activate the PRP, 23 microliters of 10% calcium\nchloride were added, followed by incubation at 37°C for\n15 minutes. After incubation, the tube was centrifuged for\n10 minutes at 4000 rpm to obtain activated PRP.\nThe assessment of volume, sperm concentrations,\nmotility, and morphology was carried out following the\nlaboratory manual from the World Health Organization\n(WHO) ( 21 ).\nThe most accurate way to determine the volume is by\nmeasuring the sample within the receptacle in which it is\ncollected. Collect the sample in a pre-measured singleuse receptacle, then measure the combined weight of\nthe receptacle containing the seminal fluid. Subtract the\nweight of the receptacle to obtain the net volume ( 21 ).\nSperm counts were conducted by observing spermatozoa\nunder a microscope using the improved Neubauer\nhemocytometer (Neubauer IMPROVED, Marienfeld,\nGermany). Sperm concentrations were calculated in\naccordance with the protocols outlined in the WHO\nlaboratory manual ( 21 ).\nThe 5 th  edition of the WHO classifies sperm motility into three categories:\nprogressive, nonprogressive, and immotile. A lower reference value of 32% is considered\nfor progressive mobility, according to the WHO directive. For the evaluation of motility,\n10 μL of liquefied sample is loaded into a clean glass slide previously maintained at\n37°C, and then covered with a 22×22 mm coverslip. The prepared sample was positioned on\nthe microscope’s heating stage set to 37°C, and it was promptly assessed under a\nmagnification of 400× ( 21 ).\nDiff-Quik staining (MICROPTIC S.L. Co., Barcelona,\nSpain) was employed to assess the impact of PRP on\nsperm morphology. The procedures were carried out\nfollowing the instructions provided in the kit. Around\n10 μl of spermatozoa was spread onto a clean glass slide\nto create a thin and even layer, which was then air-dried\nat room temperature for a minimum of 10 minutes. The\nslides were stained using the recommended staining\nprocedure outlined in the manual and examined using a\nbrightfield microscope. Typically, of 200 spermatozoa per\nsample were categorized based on their morphology, and\nthe overall number of abnormal spermatozoa was reported\nas a percentage ( 22 ).\nThe evaluation of DNA fragmentation proportion\nwas conducted using the Halo Sperm Kit (Halo kit,\nIdehvarzan Farda Company, Tehran, Iran). Initially, 50\nµL of the samples were gently combined with preheated\nagarose gel obtained from the kit, and subsequently, 20\nµL were positioned onto the previously coated glass slide.\nA 22×22 mm coverslip was then laid over the slide, which\nwas then kept at a temperature of 4°C for 5 minutes.\nFollowing this interval, the coverslip was removed, and\nthe slide was immersed in solution A (denaturing solution)\nfor 7 minutes, followed by solution B (lysing solution)\nfor 15 minutes at ambient temperature. The slide was\nrinsed using distilled water for an additional 5 minutes,\nthen subjected to dehydration through progressively\nincreasing concentrations of ethanol (70, 90, 100%).\nUpon complete drying, the slides were stained using the\nsolution provided in the kit, encompassing components\nC, D, and E, for respective durations of 75 seconds, 3\nminutes, and 2 minutes, respectively. After the staining\nprocess, a total of 200 spermatozoa were evaluated using a\nlight microscope (1000×). The presence and size of halos\ngenerated under the light microscope were indicative\nof DNA fragmentation. Spermatozoa devoid of DNA\nfragmentation exhibited sizable or moderate halos, while\nspermatozoa exhibiting fragmentation displayed minimal\nor absent halos ( 23 ).\nQuantitative clinical characteristics were described as\nmean ± SD. In this study, clinical characteristics were\nmeasured after the intervention. Mann-Whitney-U test\nwas used to compare the initial clinical characteristics in\nthe control and intervention groups. ANCOVA analysis\nwas used to compare the outcomes after intervention,\nadjusting for the initial measures of each variable and\ncovariates such as age, body mass index, duration of\nmarriage, and duration of infertility. The significance\nlevel was set at less than 0.05, and data description\nand analysis were performed using SPSS software for\nWindows (version 16.0. Chicago, SPSS inc).\n\nThe effects of PRP on sperm parameters are presented\nin Table 1 and Figure 1. The evaluation of sperm\nconcentration in the control group revealed a significant\ndecrease in the compared to average sperm concentration\nfollowing PRP injection in intervention group: 11.32 ± 8.44\nvs. 16.06 ± 15.16, P=0.030. The rate of progressive\nsperm motility in the analysis of the control group was\n8.86 ± 7.79%. Furthermore, the rate of progressive sperm\nmotility was 85.03 ± 9.64% in the intervention group. The\nevaluation of sperm progressive motility in the studied\ngroups revealed a notable rise in the average progressive motility of sperm after PRP injection compared to the\ncontrol group (P=0.014). The assessment of sperm\nmorphology revealed that the proportion of normal sperm\nmorphology in the control group was 1.63 ± 1.44%. In\ncontrast, the intervention group resulted in 1.81 ± 3.68%\nnormal morphology. However, no significant difference\nwas observed in normal morphology (P=0.628). The\nassessment of sperm morphology revealed that the\nproportion of normal sperm morphology in the control\ngroup was 1.63 ± 1.44%. In contrast, the intervention\ngroup resulted in 1.81 ± 3.68% normal morphology.\nHowever, no significant difference was observed in\nnormal morphology. However, no significant difference\nwas observed in normal morphology (P=0.628). The\nevaluation of sperm volume also showed that there was\nno significant difference between the control (2.13 ± 0.82)\nand intervention (2.24 ± 1.43) groups after PRP injection\n(P=0.663).\nComparison of sperm parameters and DFI between control (n=44)\nand intervention (n=44), after the test\nP value; The difference between control groups and intervention group. The significance\nthreshold was deemed to be P<0.05. The P value is determined by ANCOVA model. DFI;\nDNA fragmentation index, SD; Standard deviation, and ST; Statistical test.\nComparison of sperm parameters in two control and intervention\ngroups before and after the test.\nThe effects of PRP on sperm DNA fragmentation are\npresented in Table 1 and Figure 2. The percentage of\nsperm containing DNA fragmentation in the intervention\ngroup (17.23 ± 9.15%) was significantly lower than that\nin the control group (25.62 ± 12.84%, P<0.001).\nComparison of DFI in two control and intervention groups prior to\nand after the test. DFI; DNA fragmentation index.\n\nThe present study aimed to investigate the effects of\ntesticular PRP injection on sperm parameters in men\nwith severe OAT. The results of this study demonstrated\nsignificant improvements in sperm concentration,\nprogressive motility, and DFI after PRP injection.\nThe statistical analysis revealed a significant difference in\nsperm concentration and progressive motility between the\ncontrol and intervention groups after PRP injection. These\nfindings suggest that PRP treatment can improve sperm\nparameters in men with severe OAT. The increase in sperm\nconcentration and motility is particularly noteworthy, as\nthese factors are crucial for successful fertilization and\npregnancy. Concordant with our findings, Somova et al.\n( 24 ) conducted a study aligning with our research focus.\nThey observed that PRP injection in individuals with\nsevere OAT led to improved sperm concentration and\nmotility after 4 months, compared to those who did not\nreceive PRP injection. Another important finding of this\nstudy is the significant reduction in DNA fragmentation\nobserved in the intervention group after PRP injection.\nDNA fragmentation is known to have a negative impact\non sperm quality and fertility potential. The decrease in\nDFI suggests that PRP treatment may help improve sperm\nDNA integrity, which is essential for successful embryo\ndevelopment and pregnancy. The improvement of sperm\nparameters observed in this study could be attributed to\nthe growth factors present in PRP. PRP contains various\ngrowth factors, such as platelet-derived growth factor\n(PDGF) ( 25 ), transforming growth factor-beta (TGF-β)\n( 26 ), insulin-like growth factor (IGF) ( 27 ), Fibroblast\ngrowth factor (FGF) ( 28 ), and vascular endothelial growth\nfactor (VEGF) ( 29 ), among others. These growth factors\nhave regenerative and reparative properties, which can\npotentially enhance sperm production, motility, and DNA\nintegrity.\nNumerous research studies have underscored the crucial\nrole of Brain-derived neurotrophic factor (BDNF) in the male reproductive system ( 30 ), with its receptor being\nidentified within sperm ( 31 ). BDNF actively participates\nin initiating the phosphatidylinositol 3 kinase (PI3K)\npathway, potentially serving a vital function in enhancing\nsperm motility and upholding DNA integrity through this\nspecific pathway ( 32 ). As a result, any abnormalities in the\nexpression of the BDNF gene might be closely associated\nwith the onset of male infertility disorders. Moreover,\nthe expression level of BDNF in seminal fluid samples\nobtained from men with oligoasthenospermia is notably\nlower when compared to samples from infertile men ( 33 ).\nVEGF plays a crucial role as a polypeptide in the\nprocess of angiogenesis. The presence of both VEGF and\nits receptors within the male reproductive system has been\nsubstantiated, with the VEGF protein notably detected\nin spermatids, seminal plasma, Sertoli, and Leydig cells\n( 34 ). In a study conducted by Iyibozkurt et al. ( 29 ), it\nwas shown that VEGF has a beneficial effect on sperm\nmotility and linear velocity.\nPast research has showcased the existence of IGF-I\nwithin human samples, spanning across the testis,\ngerm cells, and seminal plasma ( 35 ,  36 ). Additionally,\na connection has been established between the levels\nof IGF-I in seminal plasma and the quality of semen\n( 35 ). This underscores the notion that IGF-I potentially\nbolsters sperm motility through various mechanisms ( 37 ).\nFurthermore, it is plausible that IGF-2 might contribute\nto a substantial reduction in sperm DNA fragmentation\n( 38 ). Hence, the amalgamation of these factors within\nPRP stands poised to exert a noteworthy influence on\nameliorating sperm parameters and, consequently, on\naddressing male infertility through the utilization of\ndifferent molecular mechanisms.\nIt is worth noting that no significant differences were\nobserved in normal morphology and volume between\nthe control and intervention groups after PRP injection.\nAlthough these parameters did not show significant\nimprovements, they remain crucial factors to consider in\nthe evaluation of male fertility. Further research is needed\nto explore the effects of PRP on these parameters and to\ndetermine the long-term effects of PRP treatment on male\nfertility outcomes.\nThe study’s revelations regarding the potential of PRP\nin ameliorating sperm parameters in men with severe OAT\ncarry substantial implications for both future research\nand clinical practice. While recognizing the necessity\nfor further investigation to validate these findings and\nunveil the underlying mechanisms of PRP’s impact on\nsperm parameters, the study prompts a call for more\nin-depth exploration. Future studies can probe into the\nintricate molecular pathways and growth factors involved\nin the enhancement of sperm parameters following PRP\ninjection. Additionally, the research underscores the\nimportance of ascertaining the long-term effects of PRP\ntreatment on male fertility outcomes. Subsequent research\nendeavors may focus on evaluating the sustained impact\nof PRP on sperm parameters and DNA integrity over\nan extended period, with longitudinal studies offering\nvaluable insights into the durability of the observed\nimprovements.\nIn terms of clinical implications, the study lends support to the idea that PRP holds\npromise as a safe and effective therapeutic option for men with severe OAT. This suggests\nthat PRP treatment could emerge as a compelling alternative to conventional approaches like\nhormone therapy or assisted reproductive techniques. Practicing clinicians may consider\nintegrating PRP into the treatment repertoire for male infertility, especially in cases of\nsevere OAT. Furthermore, PRP treatment provides a non-invasive and autologous avenue to\nenhance sperm parametthe sperm counters and DNA integrity. This facet carries significant\nclinical implications, offering a less invasive option for male infertility treatment.\nClinicians and patients alike may find this approach appealing due to its potential to\nimprove fertility outcomes without necessitating more invasive procedures.\n\nThe results of this study suggest that testicular PRP\ninjection can improve sperm concentration, progressive\nmotility, and DNA integrity in men with severe OAT.\nThese findings support the potential of PRP as a safe and\neffective therapeutic option for male infertility. Further\nresearch is warranted to confirm these findings and to\nelucidate the underlying mechanisms of PRP's effects on\nsperm parameters.","source_license":"public-domain-us","license_restricted":false}