Menstrual and Reproductive Function in Women with HIV-infection and Antioxidant Vitamins Deficiency

Reproductive age women proportion with HIV-infection increasing, but relationship of antioxidant deficiency and reproductive disorders in this category of patients remain unclear. Objective: To identify antioxidant vitamins deficiency and frequency of reproductive disorders interrelations in women with HIV-infection. 53 women of reproductive age with HIV-infection were divided for 4 groups according to blood serum vitamins concentration: 36 women α-tocopherol level less than 12 μmol/L; 17 women α-tocopherol level in the range 12-24 μmol/L; 35 women with retinol level less than 1.05 μmol/L and 18 women retinol level in the range 1.05-2.8 μmol/L. Spectral fluorofotometric methods were used. Statistical analysis was performed by parametric and non-parametric methods. High incidence of menstrual disorders (63%), infertility (36.1%), and luteal phase deficiency was found in the group of women with HIV-infection and α-tocopherol deficiency. Elevated levels of primary lipid peroxidation (LP) products diene conjugates, with the absence of significant differences in the content of the thiobarbituric acid reactants in this group were registered. The results obtained showed a close relationship of α-tocopherol deficiency and disorders of the reproductive system in women with HIV-infection. It is a significant reason to recommend including of antioxidant drugs in HIV-women patients of complex pathogenetic therapy to regulate adequately their reproductive function and possibly to preserve reproductive ability in these women.


Background
According to the Federal Research and Methodological Center for Prevention and Control of AIDS in the Russian Federation currently registered 246,000 HIV-infected women and 4398 HIV-infected children born by infected mothers. In the age groups 15-19 and 20-24 years there excess cases of HIV-infection among women compared with men registered since 2009. Since 2005 percentage of heterosexually acquired HIV among women is constantly growing -61%. Irkutsk Region has unfavorable epidemiological situation of HIV-infection (more than 30 thousand people), with dramatically increasing proportion of HIV-infected women, sexual route of infection prevails [1].
There are numerous studies showing increased frequency of disturbances of the menstrual cycle and ovulation [2,3], decrease in ovarian reserve [4], endocrine pathology and hypogonadism among HIV-positive women compared to HIV-negative women [5,6]. Number of ovulatory cycles and coital frequency correlate with severity of HIV/AIDS clinical status and obviously could impact fertility, and may reflect the degree of wasting and immunosuppression in AIDS [7]. Increased persistence of the human papilloma virus infection, with dysplasia and cervical cancer development [8,9], genital infections, with the further occurrence of acute or chronic inflammatory diseases of the pelvic organs [10] have been registered in women with HIV-infection. Among HIV-positive women, higher viral loads and lower CD4 counts were associated with increased cycle variability and polymenorrhea [11]. It is believed that HIV serostatus is associated with prolonged amenorrhea [2]. Highly active anti-retroviral therapy (HAART) use was not associated with prevalent abnormalities, but both HAART use and higher CD4 counts were linked to lower incidence of menstrual problems [12]. Some animal studies indicate that HAART may decrease reproductive competence. It was shown that cigarette smoking, alcohol and illicit drugs associated with a poorer response to antiretroviral therapy in women with HIV-infection [13]. Fertility and birth rates among women with HIV-1 infection are lower and the rate of early termination higher than for the general population [14].
Systemizing and identification of molecular biomarkers as reference points for the diagnosis, prevention and treatment of various disorders is of special importance [15,16]. Antioxidant defense system (AOS) is one of the key systems responsible for protecting the body from the adverse effects of both external and internal factors [17][18][19]. The most potent natural antioxidants are considered lipid soluble vitamins -αtocopherol and retinol [20]. These vitamins, being elements of AOS, prevent the development of oxidative stress (OS). Several studies show the relationship of antioxidant deficiency and the reproductive system of women, due to their involvement in the work of all parts of the regulation of pituitary gonadotrophic function [18,19]. α-tocopherol, as antioxidant effects the reproductive system state, stimulates ovaries steroidogenesis, endometrium protein biosynthesis and influence other steroid hormones synthesis target organs [19].

Objective
To identify antioxidant vitamins deficiency and frequency of reproductive disorders interrelations in women with HIV-infection

Methods
The study was performed in 2012-2014 period according to ethical standards of Helsinki Declaration (2008) in the Scientific Centre for Family Health and Human Reproduction Problems, Russian Academy of Medical Sciences (Irkutsk, Russia), Irkutsk regional infections hospital and Irkutsk regional AIDS Centre. This study was approved by the Ethic Committee of Scientific Centre of Family Health and Human Reproduction Problems (Siberian Branch of RAMS), and all involved patients signed the Informed Consent agreement for participation in the study. A random sample of women aged 18-40 years with HIV-infection from Irkutsk was carried out. The inclusion criteria: reproductive age, confirmed HIV carriers, informed consent for investigations. Exclusion criteria: excessive weight, the presence of tuberculosis, diabetes mellitus. Patients underwent interview included demographic, medical, nutritional and recreational drug-related questionnaires. A physical and anthropometrics examination performed.
Blood samples were obtained to confirm HIV, hepatitis B virus status, hepatitis C virus, to determine CD4 cell count, HIV viral load, complete blood cell count and blood biochemistry, including the serum concentrations of antioxidant parameters (α-tocoferol, retinol) and parameters of oxidative stress (plasma conjugated dienes (CDs) and thiobarbituric acid reactants (TBARs)) after overnight fasting. Lymphocyte phenotype was determined with a four-colored monoclonal antibodies immune phenotyping panel. Differential counts were determined with a cytometric method using a FacsCount (Becton Dickinson, USA). HIV viral load was obtained by reverse transcriptase-polymerase chain reaction. Patient's blood samples centrifuged for 5 min at 1.500 g at 4°C; and erythrocytes were rinsed three times with NaCl 0.9% (wt/vol). CD s absorbance detected in plasma heptanes extracts at 232 nm [22]. For conversion of absorption units to µmol/L coefficient of molar absorption (К=2.2105 М-1 С-1) was used. TBARs levels were detected by fluorometry [23] end estimated in µmol/L. α-tocopherol and retinol levels were detected in serum by fluorometry [24].
Body mass index (BMI) was calculated using the standard formula that divides weight in kilograms by the square of height in meters (kg/m 2 ).
Levels of hormones in the serum were evaluated with ELISA and radioimmunoassay devices: "Immunoassay" (Russia), Elx808 Ultra Microplate Reader Bio-Tek Instruments, ins (USA). Several instrumental methods: colposcopy, pelvic, thyroid and mammary glands ultrasound investigations on the device "ALOKA-650" were used. Human papillomavirus was detected by the method of DNA amplification or the polymerase chain reaction.
Statistical analysis was performed by STATISTICA 6.1 software (Stat-Soft Inc., USA). To compare the frequency of occurrence -using z-test or Fisher's exact test. Means and standard deviation (SD) of means were calculated and significance of differences between values was evaluated by Student (T-test) and Mann-Whitney (U-test) tests. The level of significance was set at p<0.05.

Results
Group's characteristics are shown in a Table 1. Occurrence frequency of patients with hepatitis was differences in groups 1 and 2. In group 1 HIV / hepatitis B+C combination was significantly more often -12 cases (33.3%) compared to group 2 -1 case (5.9%) (p<0.05). There were no significant differences in ages, BMI, alcohol consumption, cigarette smoking, and illicit drugs consumption (p>0.05). There were no significant differences in those who received highly active antiretroviral therapy (HAART) between groups (p>0.05).  Laboratory data are shown in a Table 2. Albumin level was significantly lower in the 1 groups (39.5 ± 5.1 g/dL) than in those who were in the 2 groups (44.9 ± 14.7 g/dL) (p<0.05). There were no significant differences in CD4 + cell counts and HIV viral loads (p>0.05). There were also no statistically significant differences between the groups in their levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), hemoglobin, plasma bilirubin (p>0.05).   Features of the menstrual and reproductive function of women surveyed are presented in Table 3. Menstrual cycle disorders were significantly more common in patients of group 1 compared with group 2 (63.9% vs. 35.3%, p<0.05). Reproductive history of women with HIV reviled high incidence of total infertility cases in group 1 (36.1% vs. 11.8% in the 2 group, p<0.05), in this case, the prevailing primary infertility -in 22.2% (p<0.05). Significant differences were recorded in the frequency of luteal phase deficiency -61.1% vs. 47.1%, p<0.05 -between the 1 and 2 groups. Significant differences in frequency of amenorrhea, oligomenorrhea, opsomenorrhea, secondary infertility and structure of the causes of infertility in group 1 compared to group 2 data were not established (p>0.05). 3 and 4 groups had no differences in both the total number of women with menstrual irregularities, and individual diseases (p>0.05). There were also no significant differences in the frequency of presence infertility and structure of the causes of infertility (p>0.05).

Discussion
It was suggested that OS may have pathological roles in development of HIV-infection [25]. It has been established that OS induces the initial stages of apoptosis, which contributes to the depletion of T cells and the progression of HIV-infection [26]. OS often occurs due to decreased activity of the antioxidant deficiency system that is typical for HIV-infection endogenous intoxication syndrome. We found primary LP products -diene conjugates increasing in women with HIV-infection and α-tocopherol deficiency. Formation and accumulation of conjugated dienes affects the permeability of membranes, and membrane-associated activity of enzymes and ion transport. This effect is the loss of membrane barrier functions which is the basis of the pathogenesis of reproductive disorders, including infertility [27]. It is believed that LP products acting on the diaphragm gametes violate its fluidity apoptotic processes in a cell and may cause infertility [28,29].
Our study proved that women with HIV and α-tocopherol deficiency have higher level of the reproductive system disorders (incidence of menstrual disorders, infertility, and a high percentage of failure of the luteal phase) than in the group with normal vitamin content. It is known that α-tocopherol affects various parts of the reproductive system and its deficiency certainly has pathogenetic importance in the development of infertility [19,30]. Thus, reduced levels of α-tocopherol may contribute to the development of reproductive disorders in women with HIV-infection.   Levels of CDs and TBARs prove the presence of oxidative stress. Table 4 demonstrates that the СDs mean level is significantly higher (1.8 ± 0.9 µmol/L) in group 1 patients than those in group 2 (1.3 ± 0.7 µmol/L) (p<0.05). No TBARs levels differences between groups registered (p>0.05) ( Table 4).
It is necessary specially emphasize the large number women with HIV/hepatitis B and C co-infection in the group with α-tocopherol deficiency. Information on combined effects of blood-borne viral hepatitis and HIV-infection on the reproductive health of women is scarce in the scientific literature [31][32][33]. Probably, the co-infection accompanying low antioxidant activity may contribute to the development of reproductive disorders in these patients and, moreover, HAART -therapy did not improve their status. We found downward trend CD4 + cells in this patients and lower albumin level due to the presence of α-tocopherol deficiency. The study also shows a tendency to viral load increase in women with HIV-infection and αtocopherol deficiency, probably due to the presence the large number women with HIV/hepatitis B and C co-infection. Association between retinol levels and frequency of the menstrual and reproductive system disorders in women with HIV-infection was not found. and α-tocopherol deficiency. It is a significant reason to recommend including of antioxidant drugs in HIV-women patients of complex pathogenetic therapy (HAART) to regulate adequately their reproductive function and possibly to preserve reproductive ability in these women. Antioxidant vitamins deficiency correction proposed to avoid reproductive disorders progressing. In this respect, further research is needed.