Diagnostic significance of Lactobacillus spp. identification in ejaculate

Popularization of the real-time polymerase chain reaction method (RT-PCR), which is a trend of the recent years, allowed to significantly expand of the range of microorganisms that can be detected in the genitourinary tract of men. Moreover, the available picture of the microbiome's bacterial component structure became more detailed. Lactobacillus spp. remains one of the least studied groups of microorganisms. Treating patients with reproductive disorders, the authors have accumulated clinical experience demonstrating the possible relationship between presence of Lactobacillus spp. in the ejaculate and changes in the level of sex hormones and the key values registered with a spermogram. This study aimed to compare the levels of luteinizing hormone, follicle-stimulating hormone, testosterone, estradiol, prolactin, progesterone, and sex hormone binding globulin (SHBG) in blood serum and changes in spermogram values in 210 men with and without Lactobacillus spp. detected in their ejaculate. The treatment group included 105 men whose ejaculate had Lactobacillus spp. in the amount of (Lg) ≥ 103, as detected by RT-PCR. The control group included 105 men whose ejaculate did not have Lactobacillus spp. detected; the microbiome's bacterial component structure of their ejaculate was normal. Compared to the control group, treatment group had hormonal disorders registered more often: abnormal levels of three or more hormones (p = 0.04), hyperestradiolemia (p = 0.05), increased level of SHBG (p = 0.01). It was established that the presence of Lactobacillus spp. in the ejaculate of treatment group participants is associated with oligoastenoteratozoospermia (p < 0.01), decreased concentration of spermatozoa (p = 0.01), their decreased motility (p < 0.01) morphology abnormalities (p < 0.01). Thus, the presence of Lactobacillus spp. in the ejaculate can be interpreted as an additional marker of hormonal imbalance and fertility dysfunction in men.

Currently, there is no consensus among the researchers as to what is the normal composition of men's urogenital tract microflora [1]. Clinical recommendations of the recent years suggest performing only the cultural analysis of the ejaculate of infertile men or preconception patients [2][3][4]. However, this method does not fully uncover the specifics of a man's urogenital microbiota: it does not allow identification of uncultivated microorganisms, e. g., obligate anaerobic and some facultative anaerobic ones, including lactobacilli [5,[7][8][9][10]. According to the leading European and Russian urologists, prostatitis diagnostics should include a two-glass test, ejaculate bacterial examination and additional RT PCR examination thereof [3,5,[7][8][9][10]. In the recent years, popularization of the advanced diagnostic methods allowed to significantly expand the range of microorganisms detected in the urogenital tract of men and women [1,8,[11][12][13]. In our opinion, one of the promising methods is RT-PCR performed with the Androflor testing kit for men; the method allows uncovering both the qualitative and the quantitative composition of the ejaculate microbiota, including lactobacilli [5,7,9,10,[14][15][16][17][18][19]. Among the published papers, there are but a few publications addressing the occurrence of lactobacilli in various biotopes of the genitourinary tract of men. According to several researchers [16,19,20], Lactobacillus spp. is one of the most common genus of microorganisms found both in healthy men and those with urethritis or prostatitis; the bacteria are identified by the bacteriological method [21], 16S rRNA sequencing [6,20] and RT-PCR [5,8,14,16,19]. Thus, Lactobacillus spp. bacteria were detected in 9-73.3% of samples of ejaculate of infertile men and those examined as part of a preconception course [5,6,8,16]. One study reported registering a statistically significant correlation between the presence of lactobacilli in the urethra and hormonal disruptors in the seminal fluid of infertile men, which is especially interesting [19].
Some papers highlight the link between the presence of lactobacilli in the ejaculate and normal characteristics of the semen as registered by a spermogram. The research showed that normal sperm morphology can coexist with an increased relative content of Lactobacillus spp. in semen samples [22]. Moreover, there was detected a positive correlation between the presence of lactobacilli in semen and normal sperm characteristics [23].
The least studied to date are Lactobacillus spp. bacteria taken from men who observed the biomaterial donation rules, i.e. abstained sexually or used barrier contraception to reduce the risk of receiving lactobacilli from a vagina. According to a number of researchers, in most cases, Lactobacillus spp. are transient microflora of a man's genitourinary tract [7,10,14,17]. Lactobacilli can play the part of a probable microbial agent that promotes emergence and persistence of a chronic inflammation of the prostate gland [24]. In the recent years, the role of hormonal changes, in particular, the effect of testosterone levels on bacterial contamination of the prostate gland secretion, has been discussed increasingly often [25,26]. However, analyzing the literature available to us we failed to discover data pointing to the correlation between the key fertility hormones -estradiol, prolactin, progesterone, and SHBG, traditionally examined in men with reproductive disorders [2][3][4], -with infectious agents identified in prostate secretions or ejaculate.
Results of a pilot study translated into a patent for an invention we obtained [27]. The essence of the discovery is that the presence of Lactobacillus spp. in the ejaculate with the bacterial titer of (lg) ≥ 10 3 can be interpreted as an additional marker of hormonal disorders and thus call for further extended examination of the man. The subject of this research is extremely relevant since there is no data describing the effect high content of lactobacilli has on the men's sperm fertility. This study aimed to compare the levels of LH, FSH, testosterone, estradiol, prolactin, progesterone, SHBG in serum and the key sperm property indicators in men with Lactobacillus spp. detected and not.

METHODS
The comparative prospective study lasted from November 2016 to July 2019 and included 210 men that visited urological clinic of the Ivanovo State Medical Academy seeking treatment for infertility, preconception course and/or having a concomitant erectile dysfunction. The inclusion criteria were: male, reproductive age; infertility or preconception preparation course; no hormonal and antibacterial drugs, as well as any other medicines, taken within the last 4 weeks. The exclusion criteria were: hypogonadotropic and hypergonadotropic hypogonadism, diabetes mellitus, hypo-and hyperthyroidism; sexually transmitted infections and clinical manifestations of prostatitis, such as pain and dysuria; karyotype abnormalities, CFTR gene mutations, microdeletions in the AZF locus of Y chromosome.
All men had their ejaculate examined with the help of the Androflor testing kit (RT-PCR); their serum was examined to determine the levels of luteinizing hormone (LH), folliclestimulating hormone (FSH), total testosterone, estradiol, prolactin, progesterone, and sex hormone binding globulins (SHBG), free androgen index (FAI), testosterone to estradiol ratio (TER), which are typically checked during examination of infertile men or men undergoing a preconception course [2,3]. Blood samples for the hormone concentration study were taken in the morning, from 8 to 10 am; the patients had to abstain from eating any food before sample taking. They were also not BULLETIN OF RSMU 3, 2020 VESTNIKRGMU.RU | | All men were divided into two groups, randomized by age, body mass index, alcohol intake and smoking status, complaints, established diagnosis and serum testosterone level (in order to exclude its influence on the bacterial content of the ejaculate) [25,26]. The treatment group included 105 men whose ejaculate had Lactobacillus spp., titer of (Lg) ≥ 10 3 , as detected by RT-PCR. The control group included 105 men whose ejaculate did not have Lactobacillus spp. detected; the microbiome's bacterial component structure of their ejaculate was normal, as registered with the Androflor testing kit. The average age of the treatment group patients was 35.5 ± 8.1 years, that of the control group patients 35.8 ± 8.3 (p > 0.05). Before biomaterial sample collection, all patients urinated, thoroughly cleaned their external genitalia (without antiseptics), and masturbated to deliver the ejaculate samples into sterile polymer containers. The containers were delivered to the laboratory within one hour from collection or less.
For RT-PCR examination, we used the DT-96 detection amplifier (DNK-Tekhnologiya; Russia) [28] and the Androflor testing kit (medical product registration certificate RZN 2016/4490 of 07. 25.2016). For the hormone concentration study we took 5 ml of venous blood from each participant.
The samples were collected from 8 am to 10 am under aseptic conditions, the blood put into 5 ml tubes. After coagulation, the liquid part was transferred to clean sterile tubes, centrifuged in a laboratory centrifuge for 10 min at 1500 rpm, then the supernatant was transferred to disposable plastic Eppendorf tubes. Hormone concentration was determined with the help of Roche Cobas e8000 602 analytical system (Roche Diagnostics; Sweden). Table 1 contains reference levels of the hormones studied. Ejaculate samples were studied with the help of the SQA-V analyzer (Medical Electronic System Ltd.; Israel). The amount of leukocytes in semen was determined by staining smears with Leukodif 200 dyes (Erba Lachema; Czech Republic); assessing the quantitative and qualitative indicators, we relied on the normal values approved by WHO (2010) [29]. Microsoft Excel 2013 and Statistica 12.0 (Stat Soft Inc.; USA) software packages enabled statistical analysis. Wilcoxon and Fisher tests were applied to determine reliability of the data obtained; the differences were considered significant at p ≤ 0.05.   23.8 11.4 3.8 Fig. 3. Changes in the hormones studied, two compared groups number of such patients was 6 (5.7%) (p > 0.05). Combined hormonal disorders were more common in the treatment group (50.5% versus 42.9%; p > 0.05) than in the control group ( Fig. 1  and 2, respectively). Men with lactobacilli had more pronounced hormonal disturbances in the form of deviations from the normal levels of three or more hormones, the difference with the values registered in the control group significant (p = 0.04). Table 1 contains the average hormone levels registered in the two groups. The group with Lactobacillus spp. had their average level of estradiol significantly different from that registered in the control group. Treatment group patients were also likely to have increased levels of prolactin and SHBG. Higher SHBG level and hyperestradiolemia were discovered in the treatment group more often (23.8% versus 11.4% at p = 0.01 and 10.5% versus 3.8% at p = 0.05, respectively), the difference with the corresponding values registered in the control group significant. As for the remaining hormones studied, there were no statistically significant differences revealed in their concentrations, however, the group with lactobacilli had a higher incidence of hyperprolactinemia (Fig. 3).

RESULTS
Compared to the control group, patients of the treatment group had their ejaculate samples more saturated with bacteria, the content measured as the total bacterial load (10 4.5 ± 0.6 versus 10 4.2 ± 0.9 , respectively, p < 0.01).
The results of RT-PCR examination (Fig. 4) show that the majority of identifiable microorganisms were registered in the treatment group more often than in the patients that had no lactobacilli detected. The difference in the incidence was significant (р < 0.05).
The results of the spermiological study (Fig. 5.) revealed that the more common types of disorder in the treatment group was oligoastenoteratozoospermia (30.0% versus 9.3%; p < 0.01) and asthenoteratozoospermia (28.8% versus 20.0%; p = 0.1). In the control group, the disorders diagnosed significantly more often were normozoospermia (42.7% versus 25.0%; p = 0.01) and isolated teratozoospermia (20.0% versus 7.5%; p = 0.01). Asymptomatic leukospermia was twice as common in the control group as in the treatment group (26.7% versus 13.8%; p = 0.03). The analysis of the key spermogram indicators (Table 2) showed that in the treatment group the motility, morphology of spermatozoids, as well as sperm concentration, were significantly worse compared to the control group.

DISCUSSION
There is an opinion that lactobacilli in men can only exist as transient flora. However, as registered in the clinical practice, some patients had Lactobacillus spp. in their ejaculate and reported over a month of sexual abstinence or strict use of barrier contraception, which minimizes the chance that such bacteria are transient in them. This research, in contrast with the pilot study [27], proved the hypothesis about the association between hyperestradiolemia and appearance of Lactobacillus spp. in the ejaculate. In the treatment group, leukospermia was a less common diagnosis than in the control group, which is probably related to the higher incidence of prostate acinus obstruction or fibrosis cases [30]. We found the ejaculate samples of the treatment group patients to be greatly contaminated with bacteria compared to those BULLETIN OF RSMU 3, 2020 VESTNIKRGMU.RU | |  of the control group (p < 0.05), which can be explained by hyperestradiolemia, increased levels of SHBG and prolactin, since the latter dampen biological activity of testosterone. One of the SHBG growth mechanisms is associated with an increased level of estradiol in blood; it often is a signal of "latent hyperestradiolemia", the latency here meaning the indicators remain within the reference range. The increased level of lactobacilli can be considered as a protectivecompensatory mechanism triggered to maintain normal ejaculate microbiome and prevent genital tract invasion with opportunistic pathogenic microorganisms [6,21,23]. In our opinion, subject to strict adherence to the rules of preparation for RT-PCR ejaculate examination, including sexual abstinence or barrier contraception for at least three days, the detection of Lactobacillus spp. in the titer of Lg ≥ 10 3 can be regarded as an additional reason to investigate the concentrations of estradiol, prolactin, progesterone and SHBG.
The analysis of the data obtained does not allow deriving whether the lactobacilli play a negative or a positive role; this may be subject of further research that, employing RT-PCR, will establish the amount of Lactobacillus spp. and determine the type of this group of microorganisms.

CONCLUSIONS
Thus, Lactobacillus spp. bacteria are more likely to be found in the ejaculate of men with hyperestradiolemia and more severe combined abnormalities as detected by the spermogram. Detection of Lactobacillus spp. in semen can be an additional marker of hormonal imbalance in men, even with the spermogram values being normal.