Menstrual Irregularities Frequency in Reproductive Age Women with Chronic Parenteral Viral Hepatitis and Insufficiency of Antioxidant Factors

Background: Our previous study of the lipid peroxidation and antioxidant defense system (LPOAOS) state in women with chronic viral hepatitis (CVH) showed the content of fat-soluble vitamin alpha tocopherol significant reduction. Reproductive health of women with CVH needs special attention. The objectives of this study was to determine the frequency of menstrual disorders in the group of women with CVH with marked alpha tocopherol insufficiency. Study Design: A case-control study. Place and Duration of Study: A study was conducted at Scientific Centre for Family Health and Human Reproduction Problems and in the hepatological department of the Irkutsk clinical hospital of infectious diseases from 2010 to 2014. This work was performed with the use of equipment of Collective research centre “Center for the development of progressive personalized technologies for health” SC FHHRP, Irkutsk. Methods and Results: Our study included 44 reproductive age women with CVH and comparable age 28 healthy women. All women underwent clinical examination and questionary survey. We found alpha tocopherol insufficiency in 95.5% cases of patients with CVH and menstrual disorders. Original Research Article Rashidova et al.; JPRI, 32(29): 60-66, 2020; Article no.JPRI.62740 61 Conclusion: Our results suggest that menstrual disorders in patients with CVH may be associated with alpha tocopherol insufficiency.


INTRODUCTION
Viral hepatitis infection with the parenteral transmission is one of the most common infection diseases, and it puts at risk people's health [1]. B, C and B+C mixed hepatitis are the most frequent to be found. One of the essential characteristics of hepatitis C is that patients develop a chronic form in 60-70% of cases [2]. Even though a chronic form of hepatitis persists only in 5-10% of patients with acute hepatitis B, the number of cases of chronic hepatitis are continually increasing. In Irkutsk region (Russian Federation) among chronic viral hepatitis infections, the chronic viral hepatitis C (HCV) takes the leading position -86.1%, and chronic viral hepatitis B (HBV) ranks the second -13.9% [3]. Chronic HCV and HBV have an overall pathologic effect on all body systems [4,5]. Reproductive health of women with these infections is of concern. We believe that determination of the major criteria to assess menstrual disorder risks in reproductive women with chronic viral hepatitis is a crucial task.
Deficit of such fat-soluble vitamins as alphatocopherol and retinol, which are antioxidants, are known to affect the reproductive system functioning. In our previous studies of lipid peroxidation and antioxidant defense system (LPO-AOS) processes we found that women with chronic viral hepatitis had marked decrease in fat-soluble vitamin -alpha-tocopherol [6,7]. Alpha-tocopherol is the main antioxidant stabilizing membrane lipid bilayer at the molecular level and provides optimal functioning of membrane receptors, systems of membrane transport, and membrane enzymes. This also includes electron transport chains determining energy storage in the cell and ATP production, and enzymes of the monooxygenase system providing biosynthesis of essential endogenous compounds such as corticoids and sex hormones [8]. Considering the significant influence of alpha-tocopherol on different parts of the reproductive system [9,10], we assessed the frequency of menstrual disorders in the group of patients with chronic viral hepatitis with marked alpha tocopherol insufficiency.

MATERIALS AND METHODS
A case-control study was conducted at Scientific Centre for Family Health and Human Reproduction Problems in the laboratories of pathological physiology, physiology and pathology of the endocrine system and in the hepatological department of the Irkutsk clinical hospital of infectious diseases from 2010 to 2014. This work was performed with the use of equipment of Collective research centre "Center for the development of progressive personalized technologies for health" SC FHHRP, Irkutsk.
We recruited women of reproductive age who then were divided into two groups according to clinical and anamnestic and laboratory data. One group comprised 44 patients with confirmed chronic HBV and/or HCV (mean age 28.9 ± 7.5) with histology activity indices, fibrosis stages, replication and integration stages (no significant changes were revealed between them), and the control group included 28 healthy women (mean age 30.8 ± 2.9).

Inclusion and Exclusion Criteria
Inclusion criteria for the group with CVH: We used blood serum, blood plasma, and hemolysate as the material for the study. Before any therapy prescribed, in the fasted state, from 8 to 9 a.m., after 15-minute rest, blood sampled from median cubital vein, using disposable vacuum blood collection tubes (on the 3-9th day of the menstrual cycle (if it is present) or in the presence of amenorrhea).
Blood was delivered to the laboratory in the periodnot exceed 60 minutes.
Chronic hepatitis was diagnosed using epidemic and clinical data. It was confirmed by specific antibodies detection by means of enzyme-linked immunosorbent assay. Intensity of inflammation was determined using histology activity index according to the classification of Knodell R.G. et al. (1981).
We conducted analysis of four menstrual cycle characteristics: regularity, cycle length, and duration and intensity of menses.
Participants of our study reported no menstrual disorders before hepatitis onset.
Intensity of lipid peroxidation processes was assessed by the blood plasmacontent of substrates with conjugated double bonds, concentration of diene conjugates, ketodienes and conjugated trieneswere measured the method based on intense absorption of conjugated dienehydroperoxide structures at 220, 232, and 278 nm. The content of TBAreactive substances was determined fluorimetrically.
We estimated the state of the antioxidant defense system by the level of the total antioxidant status (TAS), concentration of retinol and alpha tocopherol. The reference values for α-tocopherol were 7-21 μmol/L and for retinol-0.70-1.71 μmol/L.We determined activity of superoxide dismutase (SOD), concentration of reduced (GSH) and oxidized (GSSG) glutathiones in the hemolysate [11]. Measurements were made using spectrofluorophotometer Shimadzu RF-1501 (Japan).

Statistical Data Analysis
Software package STATISTICA 6.1 Stat-Soft®Inc USA was used to conduct statistical analysis. Biometric analysis included analysis of contingency tables with assessment of statistical values of one-sided Fisher's exact test.
To classify obtained data, to assess the quality of classification and to choose the most informative features we used multifactorial discriminant analysis [12]. Assessment of the frequency of antioxidant insufficiency in patients with chronic hepatitis (limits of normal values of the antioxidant defense system for groups of virtually healthy individuals) were calculated with Yates' correction (М ± t*m).
Differences of the compared values were considered significant at p≤0.05.

RESULTS AND DISCUSSION
We assessed the frequency of menstrual disorders in the group of women with chronic hepatitis. We found there were 5 types of menstrual disorders: amenorrhea (9 women), oligomenorrhea (2 women), opsomenorrhea (5 women), polymenorrhea (5 women), menorrhalgia (1 woman).
Analysis of the group of patients with chronic hepatitis showed that 50% of patients had menstrual disorders (Table 1).
Search for the most significant criteria, that would help to determine the risk of menstrual disorders development in women with chronic hepatitis was our priority task. To achieve that goal and differentiated these groups maximally we applied discriminant analysis which helped us to calculate linear classification functions depending on several most informative parameters.
We chose most informative parameters of all LPO-AOS and the neuroendocrine system regulation investigated criteria not correlating between each other to differentiate the two groups. Assessment of the parameters for their informative value was done with F-test at significance level p ≤ 0.05.
To work with the classification functions, the values were standardized according to the formula: Z = (X -m) / S, where Z is the standardized value of the variable X, m is the mean value of the variable, S is the standard deviation.
Standardization is needed to bring all parameters to consistency. Relative value of the difference between the standardized parameters exactly conforms to the relative value of the difference between the primary parameters.
To assign the tested patient to the corresponding group, the values of each parameter were entered into the equations of the classification function. The assignment of an object to a certain group is performed according to the maximum value of the classification function. If the value was positive (F1 <F2) and the woman confirmed the presence of menstrual irregularities, then the result was recognized as truly positive (a = 17 people), otherwise it was recognized as false negative (c = 3 women); If the value during testing was negative (F1> F2), and the patient did not experience any menstrual disturbances against the background of CVH, the result was recognized as truly negative (b = 19 patients), otherwise it was recognized as false positive (d = 5 women).
To determine the diagnostic significance of the selected parameters and the constructed classification equations in assessing the risk of menstrual disorders in patients with chronic hepatitis, the following parameters were determined:  Sensitivity -the proportion of patients with a positive test result among women with chronic hepatitis with confirmed menstrual disorders, -a / (a + c) × 100% = 85%,  Diagnostic efficiency of the constructed classification equations (test accuracy)the proportion of correct test results in the total number of results obtained -(a + b) / (a + b + c + d) × 100% = 81.8%.
Thus, the inclusion of these parameters into the equations of classification functions can be considered as additional diagnostic criteria that are highly sensitive and help to assign women of reproductive age with CVH in the risk group for menstrual irregularities with an accuracy of 81.8%.
Fat-soluble vitamin alpha tocopherol is one of most important components of biosynthesis and hormones functioning, regulating the reproductive system [13]. If there is alpha tocopherol insufficiency in the body the level of gonadotropic hormones significantly reduces [14]. In this connection we then conducted the Table 1

Type of a menstrual disorder
Amenorrhea(absence of menses) Oligomenorrhea(menses duration is less than 2 days) Opsomenorrhea(menses come extremely rarely: in 6 Polymenorrhea(menses' duration is 7 Menorrhalgia(the cyclic uterine bleeding, associated with menstrual cycle, lasting more than 12 days)  Table 1

. Frequency of different types of menstrual disorders in women with chronic hepatitis (%)
Proportion of the patients with chronic hepatitis (n=44), % 20.5 Oligomenorrhea(menses duration is less than 2 days) 4.5 Opsomenorrhea(menses come extremely rarely: in 6-8 weeks) 11.4 Polymenorrhea(menses' duration is 7-12 days) 11.4 Menorrhalgia(the cyclic uterine bleeding, associated with menstrual cycle, lasting more than 12 days) 2.3 frequency analysis of the prevalence of alpha tocopherol insufficiency in the studied group. Statistically significant alpha tocopherol insufficiency in 77.3% (p=0.005) of patients with chronic hepatitis (Fig. 1) was established. It may be a pathogenetic risk factor for developing disorders in a woman's reproductive system.
Analysis of the AOS state helped to determine the incidence of patients with menstrual disorders. Obtained results showed significant prevailing majority of patients with chronic parenteral viral hepatitis with marked alpha tocopherol insufficiency and presence of menstrual disorders (95.5% cases (р=0.009)) ( Fig. 2). Tocopherol influences biosynthesis of gonadotropic and steroid sex hormones by regulation of cholesterol metabolism, and contributes to normal pregnancy and normal fetus development, prevents miscarriages and disorders of sex hormones. Insufficiency of this substance leads to the uterine malfunction in women [15].

CONCLUSION
Being a general inflammatory disease, chronic viral hepatitis lead to change in the state of all organs and systems of the body including main indicators of homeostasis. In addition to inadequate cell-mediated immune response to the viral infection there are disorders in the functioning of the system LPO-AOS. Analysis of the obtained results shows changes in the functioning of the system LPO-AOS in women of reproductive age with chronic parenteral viral hepatitis. These metabolic changes can intensify the pathologic process in the future and adversely affect the whole organism. Alpha tocopherol considerable insufficiency in women with chronic hepatitis greatly contributes to menstrual disorders development because of direct influence of this component of the AOS on the functioning of the reproductive system. We may suggest that menstrual disorders in women with chronic hepatitis can relate to alpha tocopherol insufficiency. Therefore, we can recommend prescription of antioxidants, amount of which should be chosen individually considering the patients disbalance. Timely correction of antioxidant insufficiency will help to prevent the development of menstrual disorders in patients with chronic hepatitis.
Further we plan to study not only the effect of CVH on reproductive health of women, but also its influence on the course and outcomes of the pregnancy. CVH may be of particular interest to obstetrician-gynecologists because of the risk of CVH transmission to the fetus.

CONSENTAND ETHICAL APPROVAL
We conducted our study in accordance with the ethical principles of World Medical Association Declaration of Helsinki (1964, 2013 edition). Our study was approved by the local Ethical Committee of Scientific Centre for Family Health and Human Reproduction Problems. All women signed informed consent to participate in the study.

DISCLAIMER
The products used for this research are commonly and predominantly use products in our area of research and country. There is absolutely no conflict of interest between the authors and producers of the products because we do not intend to use these products as an avenue for any litigation but for the advancement of knowledge. Also, the research was not funded by the producing company rather it was funded by personal efforts of the authors.