Microbiological and clinical data associated with HPV.
Recent studies suggest that besides oncogenic capacity, HPV could have etiological role on infertility, but more evidence is necessary to confirm these results. We present in this chapter the microbiological and clinical outcome of 104 infertile women aleatory selected, from northeast of Mexico: 84.6%, with genital infection (GI) by multiple germs: Chlamydia trachomatis (Ct) [86.5%], HPV [49%], Ureaplasma urealyticum (Uu) [47.11%] and Mycoplasma hominis [35.57%]. Significant association (P ≤ 0, 05) was observed between the HPV presence and Uu diagnosis, assisted‐reproduction unsuccessful like previous treatment, cervical cytology with inflammatory process, multiple sexual partners, white‐dense‐mucous, secretion into the vagina, and HPV diagnosed in early years. The more frequent genotypes of HPV present in the infertile women studied were 6/18/16/58/11 and 68. In 60% of them, more than two genotypes were founded. The most frequent associations of high‐risk HPV (HPVhr) were 16/18, 16/58, 16/33, 16/52 and 18/58. Considering the isolate or combined presentation of HPVhr, 79.5% of these women would have a potential to develop cervix carcinoma. GI by HPV/Uu/Ct affects the fertility. Infertile women with GI that include these microorganisms with probed (HPV/Ct) or suspicious carcinogenic effect (Uu) would be considered a group of high risk for cervical cancer.
- genital infection
- Chlamydia trachomatis
- Ureaplasma urealyticum
- risk for cervical cancer
At present, reproductive failure affects to a great population. Human infertility is defined by World Health Organization, like the absent of conception and outcome of healthy baby borne, after 1 year of unprotected intercourse. Infertility is a health complex problem with low quality life for the couples. Estimations on 2010 referred 1.9 and 10.5% of primary and secondary infertility, respectively, on women at reproductive age (20–40 years old). A high prevalence of fertility health is documented in South of Asia, Medium East, Central Europe, East Europe, and Central Asia .
Genitourinary unspecific infection is associated with unexplained infertility, subfertility, obstetric and gynecologic complications that not have known clear etiology for several decades like recurrent abortion, premature delivery, placenta dysfunction and preeclampsia [2, 3]. As well, assisted reproduction treatment for infertility had been associated with genital infection [4–7]. Specific treatment for genital infection is recommended to improve the successful of
Besides the still controversial participation of HPV on fertility and obstetric complications, the major bacteria implicated for a long time are
2. Human papillomavirus
HPV belongs to
The HPV infection has an epidemiologic importance. Registered prevalence is influenced by diagnostic test applied, the number and age of individuals of studied population, the geographic region studied . Nevertheless, actually are considered the most common sexually transmitted viral infection worldwide, present in 11–12% of population and are 14 million people infected by first time each year. The incidence of HPV infection in the United States is one of higher, as 44.8%, and is present principally in women 20–24 years old . In Mexico from 2005 to 2010, urogenital candidiasis and HPV infection present an incidence of 12.3/100,000 habitant between 15 and 24 years old and were higher between 20 and 24 years old. In this country had been estimated that toward 2050, there are not a clear tendency to diminish of the HPV infection. This situation is very worrying due to the potential malignancy of the lesions . On the records of National Institute of Statistic and Geography of Mexico, were informed that 4417 women died by cervical cancer, in 2013 .
2.3. Biomedical importance
HPV infect principally undifferentiated keratinocytes into de basal level of stratified squamous epithelia, from mucous genital and oropharyngeal epithelia cutaneous and, as well as glandular cells of endocérvix [25–27]. HPV had been recognized as definitive anogenital carcinogen for male and female, mainly in uterine cervix cancer , alone or in combination with other germs . Some estimations show that the presence of HPV represents 12 more opportunities to develop cervical cancer than general population  and the HPV infection the most important between the factor risk for cervical cancer . Recently, HPV was associated, lung cancer [31–34] to larynx and pharynx carcinoma that their incidence are increasing in the last years [35, 36]. The global incidence of head and neck squamous cells and cervical cancer is similar; infection at both sites is strongly associated with sexual behavior: similarities in chromosomal aberrations, gene expression, and methylation and micro RNA profiles between Positive HPV head and neck squamous cells and cervical cancer. All of these observations were referred as argue to carry out comparative epidemiologic study of HPV infection and associated with carcinoma of head and neck and cervical cancer .
2.4. Their role in reproductive failure
Although is well known than many authors are interested to search carcinogenic roll of HPV, there are only a few studies about the effect of HPV infection on human reproduction [38–41]. There is evidence showing the adherence of HPV to the equatorial segment of sperm cell . Epidemiologic data about the infection by HPV in infertile men associated high levels of seminal leukocytes, with altered movement and morphology of head sperm, with the HPV infection [43–45]. Previously, experimental studies had been demonstrated chromosomic damage in HPV infected sperm, depending of genotypes 16 and 3 [46, 47]. An association has been found between cervical HPV of high risk and premature membrane rupture and preterm new borne [48–50]. The genital infection by more than one genotype of HPV was higher in recurrent early lost pregnancy, and HPV was identified in placenta tissue of preterm delivery by preeclampsia . The frequency of cervical HPV infection and high‐grade lesion was higher in women that have indicated assisted reproduction than general population  and successful of assisted reproduction were affected by the presence of HPV [5, 7]. The HPV transmission from mother to child
3. Chlamydia trachomatis
3.1. Biology and classification
The family Chlamydiaceae consists of two clinically important genera,
3.2. Medical relevance
4. Mycoplasmataceae Genus
4.1. Biology and classification
Mycoplasma are microorganisms derived from Gram‐positive bacteria characterized by: streamlined and very small genome, the absence of a cell wall, requiring cholesterol for membrane function and growth, and displaying genetic economy that determine a strict dependence of the host for nutrients and refuge [73, 74]. They have a parasitic lifestyle, invading target cells and existing and replicating for extended periods intracellularly . From 200 species established into class Mollicutes, six of them:
4.2. Medical relevance
Gnarpe and Friberg in early seventies informed for the first time a high percentage (85%) of infertile patients with
5. Mechanism of genital coinfection by HPV,
chlamydia trachomatisand mycoplasmas
The cervical overlaying columnar epithelia can be invaded by
6. Our experience
6.1. Studies on male infectious factor of infertility
Our group has reported different studies in couples with infertility of unknown etiology, from northeast Mexico. In that patients with spontaneous and recurrent abortion and failure in assisted reproduction treatments, was frequently detected the presence in seminal fluid, urethral swab and cervix and vagina secretions, of
A pattern of structural sperm alteration and inflammatory reaction with sperm phagocytosis, by leucocytes and macrophages, were described by semi thin section at light microscopy and thin section by transmission electron microscopy [109–113]. Beside this the infectious process result a diminish motility, vitality, linear movement and sperm concentration. These seminal parameters were associated with high levels of sperm chromatin fragmentation (SCD test), and high levels of reactive oxygen species (ROS) detected by NBT test. The antibiotic, antioxidant and drugs against the inflammation treatment determine the reduction of bacteriospermia, increase percentage of normal sperm, especially from acrosome damage, middle piece flagellum and nuclear defects. After 6 weeks of treatment, the probability of outcome of pregnancy with healthy newborn increased, and seminal parameters of predictive value were chromatin fragmentation, bacteriospermia and head sperm anomalies [114–117].
6.2. Studies on female infectious factor of infertility
After confirming that a very high percentage of couples in northern Mexico with incapacity to procreate, have
6.3. Study of HPV in infertile couples
In addition of sexually transmission of
6.3.1. Patients, material and methods
22.214.171.124. Study population
For the present analysis, samples of mucous secretion from cervix and vagina and endocervical scrapes of 104 women randomly selected women, from patients attended at private clinic in Monterrey, México, for infertility and genitourinary infection since 2003–2014. Patients were recruited by verbal invitation at the time of consultation. They gave voluntary authorization, by Informed consent, to use the clinical data and results of the laboratory test for statistical analysis. Ethical issues were in accordance with the Helsinki Declaration and endorsed by the Ethics Committee of our institution (PA15‐001). All selected patients had polymerase chain reaction (PCR) test for HPV in genital secretions and were tested by direct specimen test Kit for
126.96.36.199. Clinical assessment
An infertility medical history was taken and recorded on a standardized form by a single experienced clinician, who also performed external genital, vaginal exploration, pelvic examinations and realized the sampling of genital secretions. The data obtained from medical file were: demographic parameters, history of fertility and infertility, gestational losses and obstetric complications, treatment of infertility problems, history of cervical lesions, and surgical procedures, result of previous cervical‐vaginal cytology Pap smears, signs and symptoms of genitourinary infections and pathological data of the cervix and vagina examination. At subjective appreciation, the clinical parameters were considered for semiquantitative record: low, medium, and high or abundant.
188.8.131.52. HPV DNA extraction and genotyping
For the extraction and purification of DNA from cervical samples, 100 μL of the cell suspension previously prepared was used. Silica extraction columns (Nucleospin® Blood, Machery‐Nagel GmBH & Co., Germany) were used under the manufacturer’s instructions. During the 12 years covered by this study, the technology for HPV diagnosis was changing. For this reason, different techniques were applied depending on the time when the patient arrives for medical treatment. In 34 patients, molecular detection of HPV‐AND was carried out by PCR‐Multiplex that detect 4–12 genotypes; for 36 patients, secretions from the cervix and vagina were tested for HPV capillary electrophoresis using “Applied Biosystems 3130 series genetic analyzer systems” and the software GeneMapper, which diagnostic 14–19 genotypes high and low risk. Molecular detection of ADN from HPV was done in the 36 patients by Dual Priming Oligonucleotide, more specific and that detect 14–19 genotypes of HPV and in the 34 last patients
184.108.40.206. Statistical analysis
Absolute frequency of qualitative parameters is expressed in percentages. For quantitative variable was calculated media and, or median as well standard deviation. Sensitivity, specificity, positive and negative predictive values were calculated using (2 × 2) contingency tables. The association between clinical results and HPV and the association between other microorganisms present were calculated with Fisher’s exact test. For all tests, the significance level was P ≤ 0.05 and power of (1‐β) = 80% for two‐tail. The statistical software used was SPSS version 17 for Windows (SPSS Inc.)
From 690 infertile couples attended from 2003 to 2012, the aleatory selected sample of 104 female partners was representative, according to medium probability ponderation of 48% for the different genotypess, that was estimated based in some reports . A total of 104 female partners from couples with genitourinary infection were included in this study. Primary infertility was present in 65 couples and secondary infertility in 13 couples. The median age of the patients was 34 years (range, 22–55 years). The microorganisms identified on the group studied are
The distribution of
Between the clinical parameters taken into account for this investigation, some of them presented statistics correlation to the presence of HPV: the presence of more than one sexual partner (p = 0.05), genitourinary infection symptoms (p = 0.01), white mucous vaginal secretion fluxing from the uterine duct (p = 0.05), failure in assisted reproduction previously carried out (p ≤ 0.0001), and previous HPV infection diagnosed by suspicious lesion or cytological exam (p ≤ 0.0001) was in association to HPV (Table 1). For
|Total of cases||HPV positive||HPV negative||P|
|HPV previous diagnosis||46||44.2||33||64.7||13||24.5||0.0001|
|Pap. report: inflammation low/moderate||33||32.0||21||41.2||12||23.1||0.05|
|Active genitourinary infection||67||64.4||39||76.5||28||52.8||0.01|
|Only one partner||67||64.4||28||54.9||39||73.6||0.05|
|≥2 sexual partner||37||35.6||23||45.1||14||26.4||0.05|
|White, dense mucus secretion||78||85.7||42||85.7||36||69.2||0.05|
|Absence of vaginal microbiota||55||52.9||24||47.1||31||58.5||0.03|
|Diminished vaginal microbiota||25||24.0||17||33.3||8||15.1||0.03|
Prevalent genotypes of HPV detected in the infertile patients studied group were; HPVar 6 (19.2%), HPVhr 18 (12.5%), HPVhr 16 (11.6%), HPVhr 58 (11.1%), HPVhr 52 (7.1%), HPVlr 11 (6.7%) and HPVhr 68 (7.5%). Thirteen different genotypes of PVH were detected in low frequency. These data are concentrated on Table 2. To consider the percentage of this frequency, it is necessary to remember that the results depend on the number of HPV search in each one of the three different tests applied that was not equal for all patients.
|HPV genotype identified||Risk for cancer||Patients studied for genotype||Positive result|
Combinations of more than one HPV genotypes were detected in 60% on the studied samples. From them 46.3% were the following associations between high risk for cancer HPV: 16/18, 18/58, 16/33, 16/52 and 18/52. In a similar percentage (44.8%) of women, low risk for cancer, principally HPV 6, was detected in combination with high risk HPV: 18, 52, 16 and 39. As well, we observed that three to five different HPV genotypes were present in 23.5% of the positive group infertile patients (Figure 2).
According to the results of this study, a total of 79.5% of positive HPV group of patients, presented one or more high‐risk genotypes, and considering the statistical association between the positivity to HPV and the clinical history of previous HPV infections, this condition may represent a persistent infection or a reinfection. Consequently, we consider convenient a recommendation to take in account women with reproductive failure, like a group of risk of cervical cancer. There was statistical association between the positivity to HPV to clinical history of previous HPV infections. Would be very important, the simultaneously presence with
With the results obtained from this study, it was not possible to establish an association between the abortions presented in 28 of the studied women, and the positivity to HPV. Nevertheless for future studies that include a high number of patients, is may be important to observe that low risk to cancer genotypes 6 and 11, were present in a high prevalence in this investigation, and their importance on the results of inseminations, in vitro fertilization and gestational loss, and are not defined now a days. Finally, our data confirm the recommendation to investigate HPV before to carry out
7. Concluding remarks
This is a study carried out by aleatory selection of 104 infertile women attended by reproductive failure and genital infection; the main objective was to establish the relationship of HPV presence, with other microorganism and the genotype of HPV detected in this group of infertile women. All patients authorized the use of clinic and laboratory analysis data from her cervical and vaginal samples, to realize statistical analysis and sign an informed consent. The clinical parameters considered were: signs and symptoms of infection disease, results of previous assisted reproduction treatments, and the presence of previous abortions. These data were compared to genotype HPV present, and the diagnosis of others germs by laboratory test included direct investigation with immunofluorescence monoclonal antibodies to
In this study, more than one sexual partner (p = 0.05), genitourinary infection symptoms (p = 0.01), white mucous vaginal secretion fluxing from the uterine duct (p = 0.05), failure in assisted reproduction before carry out, and previous HPV infection diagnosed by suspicious lesion or cytological exam (p ≤ 0.0001) were in association to HPV. Prevalent genotypes of HPV detected in the infertile patients studied group were HPV six (19.2%), HPV 18 (12.5%), HPV 16 (11.5%), HPV 58 (7.2%), HPV 52 (6.7%), HPV 11 (6.7%) and HPV 68 (7.5%). Combinations of more than one HPV genotypes were detected in 60% on the studied samples. From them, 46.3% were the following associations between high risk for cancer HPV: 16/18, 18/58, 16/33, 16/52 and 18/52. In a similar percentage (44.8%) of women, low risk for cancer, principally HPV 6, was detected in combination with high risk HPV: 18, 52, 16 and 39. As well, we observed that three to five different HPV genotypes were present in 23.5% of the positive group infertile patients. According to the results of this study, a total of 79.5% of positive HPV group of patients, presented one or more high risk genotypes, and considering the statistical association between the positivity to HPV and the clinical history of previous HPV infections, this condition may represent a persistent infection or a reinfection. Consequently, we consider convenient a recommendation to take in account women with reproductive failure, like a group of risk of cervical cancer. With the results obtained from this study, was not possible to establish an association between the abortions presented on 28 of the studied women, and the positivity to HPV. Nevertheless for future studies that include a high number of patients, is may be important to observe that low risk to cancer genotypes 6 and 11, were present in a high prevalence in this investigation, and their importance on the results of inseminations,
Authors acknowledge to”Center for Research and Development in Health Sciences (CIDICS) of Autonomous University of Nuevo León”, for their assessment in molecular test for HPV, C. trachomatis and U. urealyticum, and for the financial support to this publication.