Short-Term High-Altitude Exposure (3600 m) Alters the Type Distribution of Sperm Deformity

Abstract

Background:

The effect of short-term exposure to high altitudes below 4000 m on the distribution of sperm abnormality is a matter of concern with regard to the ability for fertilization, and rarely reported.

Materials and Methods:

The survey subjects, who had not visited the high plateau previously, were divided into three groups: two high-plateau groups and the low-altitude control group. In the high-plateau groups, healthy young men had been living in Lhasa City, Tibet (3600 m), for 1 or 3 months. Similar subjects in Chongqing City (400 m) were used as the control group. Semen was collected, and semen volume, pH, and sperm concentration were analyzed. After observing sperm morphology by light microscopy, we measured the percentage of sperm abnormality and statistically analyzed its type distribution.

Results:

Sperm concentration was decreased significantly after 3 months of high-altitude exposure (p < 0.01). The total sperm malformation rate did not change, but the head malformation rate was increased (p < 0.05). In addition, there were changes in the distribution of sperm malformation. The occurrence and frequency of sperm with excessive head size, neck crimp, and tailless were increased significantly (p < 0.01, p < 0.01, and p < 0.05, respectively) at 3 months.

Conclusion:

Our study demonstrates that short-term high-altitude exposure of >1 month at 3600 m increases the distribution of sperm deformities.

Introduction

As one of the many causes of male infertility, sperm deformity has received increasingly more attention in recent years. Studies have shown a correlation between abnormal sperm morphology and the fertilization ability. The World Health Organization (WHO) proposed that, when the percentage of morphologically normal sperm is <15%, in vitro fertilization rates are reduced (WHO, 2001). Numerous studies have shown that a long-term stay of >3 months at high altitude (e.g., above 5100 m) changes semen quality (Okumura et al., 2003). However, it is unknown what happens when adult men reside at a moderate altitude for <3 months.

With economic and cultural development in China, people moving to and living at high-altitude regions (Tibetan plateau) have gradually increased. Compared with long-term exposure to high altitude, lowland-dwelling people staying at medium altitudes of 3000–4000 m for a short time are more common, such as for business trips. Large-scale personnel deployment to high altitudes occurs when troops are sent there for short-term training, drills, and rescue. One of the common features of these military personnel is that they are all low-altitude residents who have not visited a high-altitude plateau region. The effect of short-term exposure to altitudes under 4000 m on male reproduction is worthy of attention. It is necessary to first study the effect of short-term high-altitude exposure on male sperm morphology. In this study, we observed the morphological changes of sperm and the types and frequencies of sperm malformation. These data provide the foundation for further studies on the effects of short-term high-altitude hypoxic exposure on male reproduction.

In the near future, we will construct a database of sperm malformation distribution under various exposure conditions with accumulating data, and combine them with other classic injury-related indicators to establish a novel evaluation method for male reproductive pathophysiology.

Methods

Survey subjects

The survey subjects were all male Han Chinese youth born at low altitude between the ages of 18 and 24 years. They were all healthy by medical examination before going to high altitude and had not previously visited these altitudes. The subjects were divided into three groups: a low-altitude control group, a 1-month high-altitude exposure cohort (group I), and a 3-month high-altitude exposure cohort (group II). Among the subjects, those who had been living in Lhasa City, Tibet (altitude: 3600 m), for 1 month continuously (30–37 days) were used as group I (n = 4). In group II, there were 45 people who had resided for 3 months (90–97 days). For the low-altitude control group, 31 healthy men of the same age living in Chongqing City (400 m) were selected.

Medical examination

All subjects had just passed a national medical examination for new recruits, which is a thorough general medical examination on entry to military service. In particular, a physical urological examination and total serum testosterone detection were performed to exclude men with reproductive or urological diseases. In general, the urological items examined include secondary sexual characteristics and the possible presence of a varicocele or hydrocele, the location of the testes in the scrotum, and the consistency of the testes and epididymis.

Study protocol

To facilitate subject cooperation and ensure the consistency of samples, all participants were instructed to gather and provide only one semen sample. Sampling of the low-altitude group was performed in Chongqing. According to the research design, subjects in group II arrived in Lhasa City from Chongqing by railway, and after 60 days, group I arrived in Lhasa City. Thirty days later, the two groups were gathered together and semen samples were collected at the same time. For comparability, detection of all indicators was carried out by the same members of our research group.

Collection of semen samples

All subjects were instructed to abstain from sexual activity for 1 week. On the day of the experiment, they were instructed to empty their bladder, and then, sperm was obtained by masturbation. Semen was centrifuged at 1000 g for 10 minutes. The seminal pulp was collected and stored at −70°C.

Semen general physical and chemical index detection

Collected semen was thawed in a 37°C water bath and transferred to a 10 mL centrifuge tube for analysis of a series of physical and chemical indexes such as volume, pH, and concentration.

Sperm morphological observation

Semen (5–10 μL) was dripped onto one end of a slide and slowly pulled across the surface by another slide at a 45° angle to prepare a smear that was fixed and stained with hematoxylin and eosin immediately after air drying. Sperm morphology was then observed and quantitated.

Classification count of sperm deformities

Sperm was counted under a light microscope (500 sperm per sample). A deformed sperm was classified according to the location of the abnormality, namely the head, neck, and tail, and then further classified according to the morphological changes of malformation. The percentage of sperm malformations was then calculated.

Quality control

Semen analyses were usually performed twice. All deviations from some normal range of morphology were considered abnormal.

Statistical analysis

All data were analyzed using SPSS 10 statistical software. Data were first considered mean ± standard deviation, and then differences were compared by one-way analysis of variance.

Results

Table 1 shows a significant decrease in semen sperm concentration after 3 months of exposure compared with the control group and group I. The semen volume and pH did not change significantly. Figure 1 shows the common malformations of sperm after high-altitude exposure. Compared with the control, the high-altitude exposure groups showed no specific morphology under a light microscope. The total malformation frequency did not change significantly compared with the control (Table 2). However, in terms of the malformation distribution, the frequency of head malformation was increased after 1 month of exposure (p < 0.05). After 3 months of high-altitude exposure, the frequency of an excessive head size was increased significantly compared with the control group and group I (p < 0.01 and p < 0.05, respectively). However, the overall distribution of an abnormal head was not changed significantly, and was still dominated by amorphous head malformation after exposure to the plateau (Table 3).

FIG. 1.

Sperm morphology after short-term exposure to high altitude of 3600 m ( × 10). (A) Normal; (B) Excessive head; (C) Amorphous head; (D) Neck crimp; (E) Neck expansion; (F) Double tail; (G) Tailless; (H) Coiled tail.

Table 1.

Comparison of Sperm and Semen Parameters of Semen

	n	Age (year, mean±SD)	Volume (mL, mean±SD)	pH (mean±SD)	Sperm concentration ( × 10⁶/mL, mean±SD)
Control	31	20 ± 1.1	3.12 ± 1.60	7.31 ± 0.15	75.50 ± 13.66
Group I	44	19 ± 1.4	3.10 ± 1.40	7.30 ± 0.10	72.87 ± 33.09
Group II	45	21 ± 1.2	2.65 ± 1.30	7.29 ± 0.11	58.22 ± 21.91^**^,^^

p < 0.01, versus control; ^^p < 0.01, versus group I.

SD, standard deviation.

Table 2.

Comparison of the Frequency and Type of Site of Sperm Deformity in the Three Groups

	n	Total deformity (mean±SD)	Head deformity (mean±SD)	Neck deformity (mean±SD)	Tail deformity (mean±SD)
Control	31	0.38 ± 0.08	0.17 ± 0.06	0.14 ± 0.06	0.07 ± 0.04
Group I	44	0.39 ± 0.11	0.22 ± 0.09^*	0.12 ± 0.05	0.05 ± 0.06
Group II	45	0.40 ± 0.15	0.23 ± 0.09^*	0.12 ± 0.02	0.05 ± 0.03

p < 0.05, versus control.

Table 3.

Comparisons of the Types and Frequencies of Head, Neck, and Tail Deformities of Sperm in the Three Groups^a

	Head					Neck		Tail
	Excessive	Small	Double	Headless	Amorphous	Crimp	Expansion	Tailless	Short	Coiled	Double
C	0.12 ± 0.07	0.14 ± 0.06	0.06 ± 0.06	0.17 ± 0.03	0.51 ± 0.04	0.61 ± 0.19	0.39 ± 0.11	0.51 ± 0.07	0.02 ± 0.04	0.43 ± 0.05	0.04 ± 0.04
I	0.16 ± 0.10	0.15 ± 0.09	0.09 ± 0.05	0.17 ± 0.03	0.43 ± 0.02	0.61 ± 0.19	0.35 ± 0.09	0.60 ± 0.12^*	0.02 ± 0.05	0.34 ± 0.05^*	0.04 ± 0.03
II	0.22 ± 0.11^**^,^	0.13 ± 0.04	0.07 ± 0.05	0.14 ± 0.05	0.44 ± 0.02	0.82 ± 0.13^**^,^^	0.18 ± 0.12^**^,^^	0.64 ± 0.22^*	0.02 ± 0.04	0.31 ± 0.05^*	0.03 ± 0.04

p < 0.05, ^**p < 0.01, versus control; ^p < 0.05, ^^p < 0.01, versus group I.

C, control; I, group I; II, group II. All data considered mean ± SD, n = 31, 44, and 45, respectively.

Regarding changes in the frequency of neck and tail deformities, Table 3 shows that the frequency of neck crimp deformities was increased significantly after 3 months of high-altitude exposure (p < 0.01 and p < 0.01 vs. control and group I, respectively). At 1 month of high-altitude exposure, the frequency of tailless deformity was increased significantly (p < 0.05 vs. control) (Table 3).

In summary, the total malformation rate was not significantly different between plateau groups and the plain control group, but the head malformation rate was increased significantly. Further analysis showed that the type of sperm malformation was changed after short-term altitude exposure (3600 m). Specifically, the occurrence frequency of abnormal sperm with excessive head, neck crimp, and tailless was increased significantly.

Discussion

Changes in the semen concentration indicate that 3 months of exposure affect sperm production, which is consistent with previous reports (Gasco et al., 2003; Wan and Wang, 2012; Hu et al., 2016; Bai et al., 2018). This result indicates that spermatogenic cells with vigorous division rates are very sensitive to hypoxia. Another important study showed that physical exercise at high altitude can reduce sperm concentration, while sperm morphology is unaffected (Pelliccione et al., 2011).

Other parameters of semen, such as volume and pH, did not change significantly, but all of these parameters were changed and reversible in a monkey study (Saxena, 1995). One month of exposure at 3600 m increased the frequency of head deformity. It appears that the sperm head is most affected by hypoxia. Sperm development includes three important periods, 0–9, 10–14, and 70–90 days before semen ejaculation, which correspond to epididymal storage, development of sperm motility, and spermatogenesis, respectively. Interestingly, our results showed significant changes in sperm head morphology after 1 month of hypoxic exposure, suggesting that high altitude may influence late-stage sperm development, such as epididymal storage.

Head deformity results from a weak acrosome reaction induced by the human zona pellucida (Liu et al., 2003), and as a result, cervical mucus offers greater resistance to movement of the deformed sperm head, leading to infertility (Katz et al., 1990). Studies have shown that the fertilization rate of in vitro fertilization is highly predictable by observing head morphology using optical microscopy combined with the acrosome reaction (El-Ghobashy and West, 2003).

Deformities in the middle part of the sperm, such as the neck curvature and crimp, can lead to sperm inactivity or nonforward movement. Studies have shown that 5 days of short-term high-altitude exposure results in a significant reduction in the forward motor capacity of sperm (Verratti et al., 2016), which is supported by our study.

The motility of sperm is generated by swinging of the tail flagella. Malformation of the sperm tail, such as tailless or a short tail, directly affects motility. In general, a sperm tail with dysplasia of the fibrous sheath can appear as hyperplasia of the fibrous sheath, which is accompanied by the absence of central microtubules and dynein arms (Chemes and Rawe, 2003). Hypoxic exposure can increase tailless sperm, which may be comparable to dysplasia of the fibrous sheath and lead to a complete lack of motility.

The intrinsic mechanism causing sperm malformation at high altitude may be related to reversible changes in hormone levels after altitude exposure (He et al., 2015) and oxidative stress (Jefferson et al., 2004; Guthrie and Welch, 2012). At the genetic level, certain genetic polymorphisms are associated with deformed sperm (Gazque et al., 2008). For example, the PRM1-190C- > A polymorphism locus is related to male sterility in Han Chinese with deformed spermatozoa (Yu et al., 2012). Whether short-term hypoxia affects such gene loci is worthy of further study.

Limitations of our study

The sample size in our study was relatively small. Moreover, in the study design, more data are needed to prove whether the changes in morphology are a reversible process. Furthermore, the subjects were not followed up after returning back to low altitude. In terms of experimental techniques, characteristic ultrastructural changes in sperm after short-term altitude exposure need to be better assessed by electron microscopy.

Conclusion

Our study demonstrates that short-term exposure to high altitude alters the sperm concentration and frequency of sperm malformation in young men. These changes may decrease the motor function of some sperm and increase the risk of infertility. These effects, which may be reversible or the beginning of progressive sperm damage, require further analysis.

Footnotes

Acknowledgment

We thank Professor Gao Yuqi for his constructive advice on the study design.

Authors' Contributions

S.Z. carried out the study design and experimental work, and drafted the article. Y.L. and P.L. provided assistance for data acquisition, statistical analysis, and experimental work. H.T. participated in the study design and coordination of experimental work. All coauthors have reviewed and approved the article before submission.

Author Disclosure Statement

No competing financial interests exist.

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