Inexpensive thermal containers and insulation materials prevent deterioration of semen parameters for less than 90 minutes

Abstract

Male infertility contributes substantially to overall infertility, with temperature changes adversely affecting the sperm quality. During infertility treatments, the exposure of home-collected semen to extreme temperatures during transport deteriorates the semen parameters. This study investigates the effectiveness of thermal containers and insulation materials for preserving semen quality at low temperatures. Semen samples from 35 healthy male partners undergoing fertility treatments were analyzed. The samples were segregated in three groups and assessed: standard collection containers (Group A), thermal containers (Group B), and thermal containers with warming materials (Group C). Samples exposed to 4°C exhibited a notable decline in motility and forward motility over time, whereas groups B and C maintained these parameters better. Group C maintained the internal temperature at approximately 20°C for up to 90 min, reducing cold-induced deterioration. These findings demonstrate that cost-effective thermal retention methods can preserve semen quality during transport, and potentially improve the outcomes of intrauterine insemination and in vitro fertilization.

Men are solely responsible for 20–30% of the total number of infertility cases and contribute to 50% of these cases [1]. Therefore, a detailed investigation of male infertility is crucial. Oxidative stress, hormonal and anatomical abnormalities, genetic, lifestyle, and environmental factors are the major factors that contribute to male infertility [2,3,4]. Although the lifestyle and environmental factors can be controlled artificially, changes in seasonal temperatures are uncontrollable. There are a few reports on the effects of temperature changes on human sperm; nevertheless, sperm motility has been reported to decrease upon cooling [5]. When subjected to cold shock, the motility and metabolic activity of ram, bull, boar, and stallion sperm are irreversibly reduced, and the acrosome and plasma membranes are damaged [6, 7], whereas it has been reported to damage the mitochondria and reduce flagellar activity in bovine sperm [8]. Temperature variation is an important factor influencing sperm concentration and motility, thereby affecting the sperm quality [9, 10].

Semen analysis is commonly used to evaluate the sperm quality. Computer-assisted semen analysis (CASA) provides more reproducible and accurate data than conventional manual microscopy [11]. A detailed assessment of sperm parameters using CASA may be useful for predicting male fertility [12, 13]. CASA is widely used in fertility clinics and research institutions owing to its advantages of providing objective and automated analysis and reducing omissions owing to human error and subjective assessment. However, differences in the results may occur depending on the system settings and CASA type, necessitating caution in their application [13].

The motility rate of sperm reduces gradually after ejaculation, and decreases considerably in a low-temperature environment of 4°C [5]. The World Health Organization (WHO) laboratory manual recommends storing semen at a temperature close to the body temperature (20–37°C) during transport and until testing, which should be within 30 min, or 50 min at most, of collection [14]. In Japan, home collection of semen is possible. However, transporting semen to the hospitals or clinics exposes it to a hot environment of 35–40°C in summer and a cold environment of 10°C or lower in winter. Therefore, to prevent semen deterioration in winter at temperatures lower than 10°C, patients are instructed to use thermal containers or thermal insulation materials to ensure that the semen is kept warm in the container. However, owing to a lack of relevant studies, it is unclear how exposure to low temperatures affects the semen analysis results when these warming methods are used. In this study, the effects of using insulated containers and warming materials in a low-temperature environment on semen analysis was investigated, with the aim of reducing the effects that might influence the analysis.

As a preliminary experiment, the temperature inside the sperm collection vessel was measured. The study was performed at room temperature (22°C). In a cooler environment of 4°C, the temperature inside the sperm collection container in group A (collection containers) reached 20°C after 4 min and decreased to 10°C after 24 min. In group B (thermal container with collection tubes), the temperature reached 20°C after 40 min and decreased to 10°C after 2 h, whereas in group C (thermal containers with both a collection container and warming material), the temperature increased to approximately 30°C after 15 min, however, it decreased to 20°C after 95 min and to 10°C after 4 h (Supplementary Fig. 1). Therefore, a 90 min limit was imposed, as this was the maximum time for which a temperature of 20°C could be maintained. This temperature has been reported to have no effect on semen [5].

Semen analyses were performed after 30, 60, and 90 min of storage in a low-temperature environment for 35 cases, and the parameters of each time point were compared among the three groups (Table 1). After 30 min of storage, significant differences in motility, forward progressive motility rate, wobble (WOB), velocity average pathway (VAP), velocity straight line (VSL), velocity curve (VCL), and lateral head displacement amplitude (ALH) were observed between groups A and B and between groups A and C (P < 0.05) (Table 1). Linearity (LIN) was significantly different between groups A and C (P < 0.05) (Table 1). Straightness (STR) and beat cross frequency (BCF) were similar among the groups (Table 1). After 60 min of storage, significant differences in motility rate, forward progressive motility rate, STR, and ALH were observed between groups A and C, and between groups B and C (P < 0.05) (Table 1). LIN, VAP, VSL, and VCL were significantly different between groups A and B, and between groups A and C (P < 0.05) (Table 1). WOB was significantly different between groups A and B, and between groups B and C (P < 0.05) (Table 1). The BCF was similar in all the groups (Table 1). After 90 min of storage, significant differences in progressive motility rate, STR, VSL, and VCL were observed between groups A and C, and between groups B and C (P < 0.05) (Table 1). WOB significantly varied between groups A and B, and between groups A and C (P < 0.05) (Table 1). ALH levels were significantly different between groups A and C (P < 0.05) (Table 1). LIN and VAP were significantly different between the groups (P < 0.05) (Table 1). The motility rate and BCF were similar in all the groups (Table 1).

Table 1. Median values of each sperm parameter at different time points (30, 60, and 90 min) after cold exposure of raw semen.

	Raw	30 min
		group A	group B	group C
Age	32 (25–43)
Motility (%)	82 (33–98)	51 (4–81) a,c	71 (20–97) a	76 (19–95) c
Progressive motility (%)	65 (21–91)	36 (2–77) a,c	52 (15–89) a	58 (9–87) c
LIN (%)	48 (32–61)	42 (21–61) c	48 (33–70)	47 (29–69) c
STR (%)	66 (52–81)	63 (49–82)	68 (55–82)	66 (55–85)
WOB (%)	66 (51–77)	58 (42–73) a,c	64 (50–83) a	64 (45–79) c
VAP (μm/sec)	20.4 (12.2–27.6)	12.7 (4.1–23.6) a,c	20 (13.7–26.5) a	19 (11.7–32.1) c
VSL (μm/sec)	16.2 (8.6–23.1)	9.5 (2.2–18.4) a,c	15.7 (9–23.5) a	15 (9.5–27.3) c
VCL (μm/sec)	28.8 (19.7–42.9)	19.8 (9.8–39.2) a,c	27.8 (19.4–45.2) a	28.9 (17.7–43.2) c
ALH (μm)	2 (1.4–3.6)	1.4 (0.9–2.2) a,c	1.9 (0.9–3.5) a	1.9 (1.4–3.7) c
BCF (Hz)	5.7 (5.1–6.7)	5.7 (4.9–6.7)	5.8 (5–6.8)	5.7 (5.1–6.8)
	Raw	60 min
		group A	group B	group C
Age	32 (25–43)
Motility (%)	82 (33–98)	39 (3–95) c	51 (1–85) b	62 (8–92) b,c
Progressive motility (%)	65 (21–91)	24.5 (3–86) c	36 (1–72) b	45 (4–73) b,c
LIN (%)	48 (32–61)	34 (13–62) a,c	44 (22–68) a	49 (31–65) c
STR (%)	66 (52–81)	56 (41–85) c	63 (41–83) b	69 (53–80) b,c
WOB (%)	66 (51–77)	52 (29–69) a	60 (23.4–80) a,b	63 (52–75) b
VAP (μm/sec)	20.4 (12.2–27.6)	9.4 (2.8–22.4) a,c	16.1 (6.1–32.2) a	18.2 (12.2–29.2) c
VSL (μm/sec)	16.2 (8.6–23.1)	6.4 (1–18.3) a,c	13.9 (3.3–28.8) a	14.7 (8.1–26.2) c
VCL (μm/sec)	28.8 (19.7–42.9)	17 (9.1–35.6) a,c	23.4 (3.3–42.6) a	25.3 (17.8–43.1) c
ALH (μm)	2 (1.4–3.6)	1.3 (0.9–2.6) c	1.6 (0.8–5.9) b	1.7 (1.2–3.2) b,c
BCF (Hz)	5.7 (5.1–6.7)	5.6 (4.5–7.1)	5.7 (5–7.1)	5.6 (5.1–6.3)
	Raw	90 min
		group A	group B	group C
Age	32 (25–43)
Motility (%)	82 (33–98)	36 (4–64)	34 (7–83)	49 (1–89)
Progressive motility (%)	65 (21–91)	24 (1–48) c	25.5 (1–59) b	33 (1–70) b,c
LIN (%)	48 (32–61)	29 (11–49) a,c	36 (6–54) a,b	41 (17–72) b,c
STR (%)	66 (52–81)	54 (34–73) c	60 (16–77) b	64 (26–86) b,c
WOB (%)	66 (51–77)	50 (6.2–63) a,c	56 (38–68) a	58 (6.3–80) c
VAP (μm/sec)	20.4 (12.2–27.6)	8.8 (4–14.3) a,c	12.6 (4.8–21.6) a,b	14.6 (6.2–26.9) b,c
VSL (μm/sec)	16.2 (8.6–23.1)	5.4 (1.7–27) c	9.6 (0.6–18.6) b	11.3 (2.5–24.7) b,c
VCL (μm/sec)	28.8 (19.7–42.9)	15.9 (10.7–28.3) c	18.7 (10.1–33.5) b	21.5 (12–38.8) b,c
ALH (μm)	2 (1.4–3.6)	1.3 (0.9–2.6) c	1.3 (0.7–4.1)	1.5 (0.9–2.8) c
BCF (Hz)	5.7 (5.1–6.7)	5.6 (4.6–8.0)	5.5 (4.2–6.8)	5.4 (4.5–6.5)

a: A vs. B, b: B vs. C, c: A vs. C. ^abc P < 0.05.

The effects of different storage methods on changes in parameters over time were analyzed for each group (Fig. 1). The motility (Fig. 1A) and forward progressive motility (Fig. 1B) rates decreased over time in all the groups. Furthermore, significant differences were observed between the RAW and the 30-min samples for group A, 30- and 60-min samples for group B, and 60- and 90-min samples for group C (P < 0.05). These findings indicate that thermal containers and warming materials can preserve motility and forward progressive motility rates. LIN decreased significantly in groups A and B between RAW and the 90-min samples and between the 30- and 90-min samples (P < 0.05); however, group C exhibited no significant decrease and was preserved until 60 min (Fig. 1C). Group C did not exhibit a significant decrease in the STR (Fig. 1D) or WOB (Fig. 1E) over time. Parameters including VAP (Fig. 1F), VSL (Fig. 1G), VCL (Fig. 1H), and ALH (Fig. 1I) were significantly lower in the samples of group A than in the RAW samples, and after each elapsed time (P < 0.05). The BCF differed significantly only between the 30- and 90-min samples in group B (P < 0.05) (Fig. 1J). Overall, with the exception of the BCF, B and C effectively preserved the parameters for up to 90 min.

Fig. 1.

Semen analysis after 30, 60, and 90 min of exposure to three different methods in a low-temperature environment. Three semen samples were prepared from groups A, B, and C. All thermal containers were incubated at 4°C for 30, 60, or 90 min and the semen parameters were measured (n = 35). The effects of different storage methods on the analyzed parameters were compared using semen analyses at 30, 60, and 90 min. The following parameters were analyzed: (A) motility rate, (B) progressive motility rate, (C) linearity (LIN), (D) linearity (STR), (E) wobble (WOB), (F) velocity average pathway (VAP), (G) velocity straight line (VSL), (H) velocity curve (VCL), (I) lateral head displacement amplitude (ALH), and (J) beat cross frequency (BCF). Group A was marked in blue, group B in orange, and group C in green. * P < 0.05.

This study shows that semen degrades relatively quickly when exposed to 4°C. Sperm viability, motility, and forward motility rates decrease substantially over time when exposed to low temperature environments [15, 16]. Low-temperature (below 10°C) environments inhibit sperm metabolism and mitigate death, however, exposure to rapid cooling causes “cold shock,” which reduces sperm motility and viability [17]. Reactive oxygen species (ROS) generated in low-temperature environments damage the mitochondria and membranes [18, 19]. Mechanistically, cold shock is linked to phase changes in lipids in the spermatozoal membranes [20]. However, abrupt lipid phase transitions have not yet been detected in humans [21].

Parameters that affect motility include VCL, VSL, and VAP [22, 23]. As velocity parameters such as VSL, VCL, and VAP decrease, STR, LIN, and WOB are not expected to change significantly because of the relationship between these parameters (STR = VSL/VAP; LIN = VSL/VCL; WOB = VAP/VCL). The median VSL, VCL, and VAP values decreased significantly over time. Changes in STR, LIN, and WOB were observed over time; however, the decreases were more gradual than those for VSL, VCL, and VAP (Fig. 1). In a study on cat sperm stored at low temperatures, the BCF was reported to decrease significantly from day 7 [24]. In this study, no significant difference was observed because of the relatively short exposure (less than 90 min) to the low-temperature environment. However, a significant difference was observed when exposed for a longer period (7–10 days). There are a few reports on the effect of temperature on BCF, wherein BCF remains unaltered over the course of a day in cold environments. Therefore, BCF may not be an important parameter to consider in assisted reproductive medicine, where semen is processed on the day of collection.

Forward progressive motility and the number of sperms in the insemination fraction are important factors with respect to the male side for the success of intrauterine insemination [23, 25]. Sperm motion parameters are closely related to semen fertility and quality [26]. Therefore, maintenance of motility is of critical. To prevent the deterioration of semen parameters owing to the low-temperature environment, a heat source was placed inside the warming container to provide greater heat retention than that achieved when only the warming container was used. Although previous studies have examined rabbit and dog semen stored at low temperatures (5°C) [27, 28] and the effects of body temperature and room temperature storage on human semen parameters [5, 15,16,17,18], reports are still inadequate on the methods for preserving human sperm at low temperatures without adverse effects. In this study, we demonstrated that the use of additional warming material inside a warming container in a low-temperature environment can prevent the deterioration of human sperm parameters such as motility and forward motility rates, albeit for a limited duration of 90 min. To the best of our knowledge, this is the first study to report the effectiveness of a low-cost, simple, and commercially available warming container and warming material in inducing a temporary temperature increase.

The warming material used in this study, microwaved at 500–600 W for 10 sec, temporarily increased the temperature inside the warming container to less than 30°C, above body temperature, without causing adverse effects. Further heating of the warming material may keep the product warmer for a longer period; however, the material used in this study ruptured when heated beyond 10 sec, indicating that this was the upper temperature limit for safe use. Moreover, placing a heat source at an extensively high temperature in a warming container can negatively affect semen quality. Our clinic advises that specimens be brought within 3 h of collection. Therefore, development of a more effective method to maintain a temperature of 20°C or higher for at least 3 h is crucial. Notably, this study was performed in a simulated cold environment, rather than at actual winter temperatures. In real-world scenarios, it is unlikely that semen would be constantly exposed to temperatures as low as 4°C, which indicates a limitation of this study. Furthermore, semen was dispensed into 1.5-milliliter tubes to accurately measure the effect of temperature change on semen attributes in this study. The thickness of the tubes may have mitigated the decrease in temperature. It should also be noted that because semen is usually transported in volumes greater than 1 ml, the fluid volume may have mitigated the temperature change.

In conclusion, the use of a warming container and warming material prevented the deterioration of sperm parameters, albeit only for a short period (approximately 90 min). In particular, the addition of inexpensive warming materials to the warming container enabled maintenance of the internal temperature for longer than possible with the warming container alone, preventing the deterioration of sperm parameters.

Materials and Methods

Ethical approval

Semen samples used in this study were collected from 35 healthy male partners of female patients who had undergone infertility treatment at our university hospital between January 2022 and April 2024. Only the semen samples determined to be “healthy” according to the WHO guidelines (2021) were used for analysis. Informed consent was obtained from all the participants who donated their semen. This study was performed in accordance with the guidelines of the Ethical Review Committee for Clinical Research of Mie University Hospital (approval number: H2021-259).

Preliminary investigation

Collection containers (SARSTEDT, Nümbrecht, Germany), thermal containers (SEED POD; TENGA Healthcare, Tokyo, Japan), and disposable warming materials (Snowpack Puchi; Mie Chemical Industry, Mie, Japan) for carrying the collected sperm were sorted into three groups: Group A, sperm collection container; Group B, sperm collection container and thermal container; and Group C, sperm collection container, thermal container, and warming material. A SmartButton Starter Pack (ACR Systems, Surrey, BC, CA) was placed in the sperm collection container, and the temperature was measured with the lid closed. The materials were warmed in a microwave oven at 500–600 W for 10 sec, placed on the lid of a collection container stored in a thermal container, and the lid was closed. The materials in the three groups were placed in a static environment at 4°C, and the change in the temperature inside the container over time was measured. Preliminary data were used in this study.

Study design

Samples (200 µl) remaining after semen testing were dispensed into nine 1.5-milliliter microtubes (Supplementary Fig. 2). The microtubes containing 200 µl of semen were placed in collection containers, which were then placed in their own thermal containers. Three thermal containers were constructed for each group (A, B, and C). All the thermal containers were incubated in a refrigerator (PHCbi, Osaka, Japan) at 4°C for 30, 60, and 90 min, respectively, and the semen analysis was performed (n = 35).

Semen analyses

Samples with a total semen volume of less than 2.0 ml, a sperm concentration of less than 5.0 × 10⁶/ml, and motility of less than 30% were excluded from the analyses. Semen samples were liquefied for approximately 30 min after collection. The semen volume was measured and analyzed using a CASA system (LensHooke X1 PRO; Bonraybio, Taichung, Taichung, Taiwan) [12, 29]. The CASA system is an integrated measurement and analysis system that does not include an eyepiece and uses a dedicated cassette instead of a slide. The depth of the measuring part of the cassette was 2 µm. For each group, all the samples were then exposed to 4°C for 30, 60, and 90 min and subsequently subjected to CASA (n = 35). The fluid volume, sperm concentration, motility rate (total and forward motility), and morphology were measured; parameters measured using CASA included semen concentration, sperm count, motility rate, forward progressive motility rate, motility sperm count, forward sperm count, VAP, VSL, VCL, VAP (≥ 25 μm/sec), VAP (<25 μm/sec), LIN, STR, WOB, ALH, BCF, and sperm morphology.

Statistical analysis

Data are presented as median values. Each value was analyzed using a nonparametric test. Friedman test was performed for all the cases and adjusted using the Bonferroni method. Statistical significance was set at a P-value < 0.05. Statistical analyses were performed using SPSS Statistics ver. 29 software (IBM Corp., Armonk, NY, USA).

Conflict of interests

This study was performed in collaboration with TENGA Healthcare, which provided funding and resources to support this study. TENGA Healthcare had no influence on the study design, data collection, analysis, or interpretation of results.