Evaluation of the effect of Platelet-Rich Plasma (PRP) on the sperm parameters

Abstract

Objective

Platelet-rich plasma (PRP) has been widely used to treat women with infertility but is rarely used in men with similar conditions. Several studies have shown that PRP contains a large spectrum of growth factors that may benefit sperm, but the application of PRP in sperm processing techniques has not been examined completely yet. This study aimed to investigate the effect of adding PRP supplementation to human semen in the incubation and swim-up processes and evaluate the sperm parameters after PRP addition.

Material and method

The current experimental study has included 80 semen samples. The semen samples were collected from 40 men with asthenoteratozoospermia and 40 normozoospermic men. The study was conducted at the Infertility Center of Taleghani Hospital from October 2023 to April 2024, and the role of PRP on sperm parameters was tested based on WHO guidelines. Each sample was divided into two equal parts. The first one, as a control, received no platelet-rich plasma, and the second sample portion was added directly to 2 % concentrations of platelet-rich plasma. The incubation and swim-up processes were performed in two groups, and sperm parameters were compared between samples incubated with PRP and samples without PRP treatment.

Results

After incubation, the results of normal samples showed a significant rise in sperm total and progressive motility. However, the increasing effect on sperm non-progressive motility and viability was not substantial. The asthenoteratozoospermic samples showed a significant rise in sperm total, progressive, and non-progressive motility, but the increasing effect on sperm viability was not significant. After swim-up, the results of normal samples showed a significant rise in concentration and sperm total and progressive motility but the rise in sperm non-progressive motility and morphology was not significant. The results in samples with the asthenoteratozoospermic group showed a significant rise in concentration, total motility, progressive motility, and non-progressive motility but the rise in sperm morphology was not significant.

Conclusion

These results show that PRP has effectively improved human sperm quality. So we can suggest that incubation and swim-up of semen samples with PRP may be considered as a new treatment option for men with infertility undergoing assisted reproductive technology (ART).

Introduction

Infertility is characterized as a condition in which couples fail to conceive after engaging in unprotected sexual intercourse for a year [ ]. The prevalence of infertility ranges from 8 to 15 %, with male factors accounting for approximately half of these cases [ ]. Male infertility, which can significantly impact infertile couples, may stem from various factors, including sperm dysfunction [ ]. Abnormalities in sperm parameters, such as low concentration, reduced motility, or high levels of abnormal morphology, are considered crucial factors in male infertility and can diminish the success rates of ART procedures [ , ]. Traditionally, male infertility and sperm quality are evaluated through microscopic analysis of sperm concentration, motility, and morphology. However, this examination method is insufficient as a diagnostic tool for male infertility [ ]. Despite its limitations in accurately predicting male fertility and ART success, this approach remains widely utilized [ ]. Previous research has indicated that nearly 30 % of male partners with seemingly normal sperm parameters are unable to conceive, highlighting the potential shortcomings of routine analysis as a diagnostic tool [ ].

Recently, platelet-rich plasma (PRP) has gained prominence as a novel treatment option for infertility. PRP is defined as a concentration of autologous or allogeneic human platelets that is 3–8 times higher than its normal concentration in whole blood, typically around 2 % [ , ]. The therapeutic benefits of PRP are attributed to the contents of alpha granules and bioactive components found in blood platelets, which actively promote cell proliferation and differentiation [ , ]. These alpha granules contain various growth factors, including transforming growth factor b, fibroblast growth factor, vascular endothelial growth factor, insulin-like growth factor 1, platelet-derived growth factor, and zinc, all of which contribute to its potential positive effects on human sperm quality [ ]. Vascular endothelial growth factor (VGEF) has been demonstrated to enhance sperm motility in vitro, while insulin-like growth factor I (IGF-I) improves sperm motility, viability, and the integrity of mitochondrial and plasma membranes [ ]. Additionally, incubation with fibroblast growth factor 2 (FGF-2) has been found to increase the progressive motility and kinematics of spermatozoa [ ]. A recent study examining the role of PRP in sperm physiology demonstrated that sperm samples incubated with PRP for 5 min exhibited significant improvements in motility and morphometry [ ]. While numerous studies have explored the effects of PRP on the female reproductive system, fewer have investigated its impact on men with similar fertility issues. Although some research suggests that PRP growth factors may enhance sperm quality, the specific effects of incorporating PRP into the incubation and swimming processes on sperm quality have not been thoroughly examined. Consequently, this study aims to investigate sperm parameters in PRP-incubated and PRP-swam samples, focusing on concentration, motility, morphology, and viability in infertile male couples [ , ].

Materials and methods

In this study, a prospective design was conducted at the andrology laboratory of the Infertility Center of Taleghani Hospital, Tehran, Iran, from October 2023 to April 2024. The participants were 80 men (40 men with asthenoteratozoospermia and 40 normozoospermic men), aged 20–45 years, who were referred to Taleghani Hospital Reproductive Center for infertility evaluation and had asthenoteratozoospermia or were normozoospermic in their semen analysis. The study was approved by the author’s institute ethics committee at Shahid Beheshti University of Medical Science (IR.SBMU.RETECH.REC.1402.753). Each participant had given informed consent before being included in this trial.

Collection and preparation of sperm samples

The participants of the study were selected from 40 normozoospermic and 40 asthenoteratozoospermic men who came to the andrology laboratory for fertility evaluation. After excluding patients with alcoholism, drug addiction, smoking, urinary tract infection, chronic disease, and any systemic disease, informed consent was obtained from all subjects before inclusion in the study. All participants had a normal body mass index (BMI). This experimental study was conducted on 80 sperm samples. Patients were asked to collect a sperm sample in a sterile plastic or glass container by masturbation after 2–7 days of sexual abstinence. Samples were placed in a 37 °C incubator for approximately 30–60 min to complete liquefaction. Samples and semen parameters were then routinely analyzed according to the WHO 2021 standards.

Macroscopic characteristics, such as liquefaction time, volume, appearance, and viscosity, and microscopic characteristics, including sperm count, motility, and morphology, were assessed by a single technician. After analysis of sperm parameters, each sperm sample was divided into two equal parts. The first received PRP at a concentration of 2 %, while the second was not. After approximately 1 h of incubation at room temperature, sperm samples were subjected to routine sperm analysis based on WHO 2021 to assess sperm concentration, motility, and morphology. We then compared the PRP-incubated group with the untreated group in terms of sperm parameters.

Based on previous studies and after using several different concentrations of PRP (2 %, 5 %, and 10 %), it was found that 2 % is the optimal percentage to make PRP and produces significant effects with remarkable results [ , ]. Also, our study showed that platelets at a concentration of 2 % can improve the progressive motility of sperm. The results are consistent with Bader et al. So the protective concentration of PRP was 2 % in this study, and the concentrations at 5 % and 10 % were not efficacious [ ].

After evaluating the effect of PRP addition on the incubation process, we investigated the role of PRP in the swimming technique of sperm production.

The swim-up technique is a method used to separate motile sperm from nonmotile sperm in a semen sample. In this process a layer of culture medium is placed over the semen, and motile sperm swim into the medium, leaving the nonmotile sperm behind. This technique is a simple step in in vitro sperm selection.This method accumulates the most motile sperm in the upper fraction and leaves sperm with low or no motility in the lower fraction. One limitation of this technique is the presence of immature germ cells, dead spermatozoa, and cellular debris. Consequently, these elements may be misidentified as immotile spermatozoa [ , ].

In this study, semen samples were kept in an incubator for 20 min during which liquifaction was completed. Following this, the sperm preparation process was initiated. 5 μL of semen was taken from the sample in order to assess sperm count and motility prior the preparation process. After the microscopic evaluation of the sample, the remaining semen was washed with the “swim-up” technique.

In standard swim-up technique, following liquefaction, 1 ml of whole semen was gently mixed with 1 ml of Ham’s F10 medium, then centrifuged (at 900 rpm for 10 min). The supernatant was removed with the aid of a micropipette and 2 ml of “Ham’s F-10” media was added again to the pellet, then incubated for 1 h at 37 °C. A sample was again taken from the supernatant and the sperm concentration, motility, viability and morphology were analyzed.

Thus, one group was treated using the routine swimming method, where the sperm was centrifuged and separated from other seminal components, and the other group was treated with PRP components using the swimming method.

Preparation of PRP

On the day of sperm collection and after informed consent, blood samples were taken from volunteers to prepare PRP. We collected samples of five volunteers for blood donation. They were healthy men with an age range between 20 and 45 years. Collected intravenous blood was processed to yield PRP. Non-autologous PRP was prepared according to the manufacturer’s instructions (Gena Medical, Tehran, Iran). In this way, approximately 10–20 ml of venous blood samples were transferred to a sterile falcon tube containing an anticoagulant solution and centrifuged twice. The first centrifugation was performed at 150 g for 10 min. The blood was separated into three layers: the supernatant, the middle Buffy coat, and the red blood cells. The supernatant and interlayer were transferred to a new centrifuge tube and centrifuged again. A second centrifugation was performed at 1200 g for 15 min to separate platelets from the blood and obtain PRP. About three-quarters of the supernatant was discarded and the retentive supernatant was counted as PRP. This PRP product was aspirated and transferred into a sterile falcon tube.

Evaluation of sperm parameters

Sperm morphology : was evaluated with Diff Quick staining. Briefly, the slides were fixed for 15 s with fixative before being stained in Diff Quick Solution I for 10 s. Finally, slides were flooded by Solution II for 5 s. Instantly, the excess stain was removed by washing in distilled water, and the smears were allowed to air-dry.

Sperm motility: Sperm motility was assessed by counting 200 spermatozoa per sample. The samples were divided into three categories: progressive motility: when sperms actively move fast and slow in linear or circular motions; non-progressive motility: when the sperms are moving in a circle or tail motion is observed; immotile: no movement is noticed (WHO 2021).

Sperm viability: Eosin-nigrosin staining was used to evaluate sperm viability. Briefly, 10 μL of each sample was mixed with dye for 30 s, and the mixture was immediately smeared onto a glass slide. Smears were prepared by adding 10 μL of mixture droplets dripped on the slides. After air drying, a minimum of 200 sperm cells were counted with a microscope. Unstained sperm were classified as viable, and the dead cells were stained red. The proportion of dead and live spermatozoa was determined based on observing at least 200.

Statistical analysis: The data obtained from this research was expressed as mean ± SD and was analyzed statistically using SPSS version 24 software. A P-value of 0.05 or less was considered statistically significant.

Results

Our findings about the comparison of normospermic semen samples after incubation with and without PRP are shown in Table 1 . The results showed that in 40 normal semen samples, incubation with PRP for 1 h yielded a better sperm quality than the other group. Adding PRP improved sperm quality in terms of total motility (65.2 ± 13.2 % vs. 89.8 ± 8.9 %, p < 0.05), progressive motility (55.2 ± 12.9 % vs. 77.8 ± 14.2 %, p < 0.05), non-progressive motility (10.2 ± 0.4.2 % vs. 11.1 ± 4.2 %, p > 0.05), and viability (82.5 ± 10.5 % vs. 83.9 ± 6.4 %, p > 0.05). There was a significant rise in sperm total and progressive motility, but the increasing effect on sperm non-progressive motility, and viability was not significant. Here in, we showed that although PRP improved sperm motility, but it did not effect on sperm concentration and morphology in normospermic semen samples.

Table 1
Comparison of sperm parameters after incubation with and without PRP in normozoospermic semen samples.
Sperm Parameters Incubation (Mean ± SD) n = 40 Incubation + PRP (Mean ± SD) n = 40 P-value
Concentration (1 × 10 6 /mL) 34.5 ± 5.3 34.5 ± 8.2 >0.1
Total motility (%) 65.2 ± 13.2 89.8 ± 8.9 <0.001
Progressive motility (%A + B) 55.2 ± 12.9 77.8 ± 14.2 <0.001
Non-progressive (% C) 10.2 ± 4.2 11.1 ± 4.2 0.344
Viability (%) (Eosin test) 82.5 ± 10.5 83.9 ± 6.4 0.476
Morphology (%) 4.5 ± 3.2 4.5 ± 5.4 >0.1

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May 11, 2025 | Posted by in OBSTETRICS | Comments Off on Evaluation of the effect of Platelet-Rich Plasma (PRP) on the sperm parameters

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