EMERGING REPRODUCTIVE HORMONES PERTURBATIONS IN HIV POSITIVE FEMALES: A NORTH INDIAN STUDY

Asha Kumari 1 , Shashi Seth 2 , Uma Chaudhary 3 , Veena Singh Ghalaut 4 and Piyush Bansal 5 , Manish Raj Kulshreshtha 5 . 1. Demonstrator, Department of Biochemistry, PGIMS, U.H.S. Rohtak, Haryana, India, 124001. 2. Ex-Senior Professor, Department of Biochemistry, PGIMS, U.H.S. Rohtak, Haryana, India, 124001. 3. Senior Professor & Head, Department of Microbiology, PGIMS, U.H.S. Rohtak, Haryana, India, 124001. 4. Senior Professor & Head, Department of Biochemistry, PGIMS, U.H.S. Rohtak, Haryana, India, 124001. 5. Assistant Professor, Department of Biochemistry, BPS GMC for Women, Khanpur Kalan, Sonepat, Haryana 6. Assistant Professor, Department of Biochemistry, RML, Lukhnow, Uttar Pradesh.


Introduction:-
Chronic complications of HIV infection in females unveils in form of menstrual abnormalities, early menopause, lower genital tract neoplasias, sexually transmitted infections, cardiovascular complications and osteoporosis apart from fertility and longevity issues. (1,2) HIV infection affects endocrinal glands by direct and indirect mechanisms. Data accruing suggests that HIV positive females get diseases of aging at younger age with increased severity than uninfected ones. They lose ovarian function at earlier age also bringing menopause prematurely.(3) Management of HIV positive females can be improved substantially if endocrinal derangements are detected in advance. Very few studies have been done in India to understand the profile of endocrine dysfunctions in HIV patients. The present study was undertaken to understand the endocrine involvement in HIV seropositive females, and an effort was also made to correlate these endocrine abnormalities with stages of HIV infection and CD4 cell counts.
Aim and objectives:- To estimate the hormone levels: Testosterone, Estrogen and Progesterone in 47 HIV seropositive females and age matched controls  To correlate these hormone levels with CD4 cell counts.

Subjects and method:-
This observational cross sectional study was conducted with ethical clearance in the Department of Biochemistry in collaboration with Department of Microbiology; Pt. B.D. Sharma Post Graduate Institute of Medical Sciences, Rohtak, Haryana, India. Forty seven known female clients of HIV infection coming to Integrated counselling and testing center (ICTC) for diagnosis of HIV infection and those attending ART center for CD4 monitoring, were enrolled along with forty seven age matched controls on giving an informed written consent after full explanation of the purpose and nature of all procedures used.
Three serological rapid tests based on three different principles diagnosed a case. CD4+ cell counts were done by flow cytometry. Serum estrogen, progesterone and testosterone were estimated on ADVIA Centaur CP by a competitive immunoassay using direct chemiluminescent technology.(4,5,6) Cases were divided into three groups on the basis of baseline CD4 cell counts as: • Group A-CD4 cell counts < 200/mm3 • Group B-CD4 cell counts 200-350/mm3 • Group C-CD4 cell counts > 350/mm3. Samples were taken for routine investigations and hormones including Estrogen, Progesterone and Testosterone on the second day of menstrual cycle.

Sample collection and storage:-
Fasting early morning venous blood sample was taken in a plain red capped evacuated blood collection tube under all aseptic precautions. Samples were processed within one hour of collection. Serum was separated by centrifugation at 2000 rpm X 10 minutes after clotting. Separated serum was stored at -20•C (maximum 3 months) for evaluating hormone levels.
Statistical analysis:-Data were presented in mean (SD), median and percentage. IBM SPSS ver. 20 was used for various statistical analyses. Comparison of data between groups was done using't' test / Mann Whitney Test for quantitative data and Chi-square test for qualitative data. Comparison between multiple groups was done using one-way anova/ Kruskal 70 wallis test. Paired samples were compared by paired't' test / Wilcoxon sign test. Correlations and regression between groups were analyzed using suitable models. Charts and graphs were prepared using IBM SPSS ver. 20 and Microsoft excel programs.

Results and observations:-
Mean age in female cases was 29±7 years with a range between 17-41 years. On the basis of CD4 cell counts cases were divided in group A (<200 cells/mm3), group B (200-350 cells/mm3) and group C (>350 cells/mm3   Previous studies had also reported that significant androgen deficiency is common among women with HIV disease (7,8) occurring in 66% of cases with wasting.(9) Grinspoon et al reported that mean free testosterone, but not total testosterone levels were decreased in subjects with wasting compared to those in age matched healthy controls. (9) So women loses significant lean body and muscle mass in late stages of wasting. However, in contrast to men, women exhibit a progressive and disproportionate decrease in body fat relative to lean body mass at all stages of wasting, consistent with gender-specific effects in body composition in Acquired Immunodeficiency Syndrome wasting. (9) Cofrancesco et al found decreased testosterone levels and increased Sex Hormone Binding Globulin (SHBG) in HIV-infected women with CD4 cell count <200cells/mm 3 (10) Another study noticed that serum total (P < 0.0001] and free testosterone levels (P < 0.0001) were significantly lower in HIV-infected women (n = 37) than in healthy women (n = 34). Testosterone levels were significantly lower in infected females even if they were menstruating normally and were independent of weight loss (11). Our second finding was lower serum estrogen levels in cases than controls. (Table 1, Fig 2) Correlation was found between estrogen and CD4+ cell counts and estrogen with BMI. (Table 2) In contrast, Estrogen levels were not different between HIV positive female cases and controls in study of Jain et al, and this result was not affected in a secondary analysis adjusting for Sex hormone binding globulin. But estradiol levels were lower among subjects with amenorrhea compared to eumenorrheic HIV-positive and controls and did not correlated with body composition (13).
72 Weinberg et al on the other hand studied sex steroid levels in 20 HIV infected and 20 HIV uninfected nonpregnant, not receiving exogenous sex steroids and women with regular menstrual cycles at three time points after menses and reported that the HIV infected group had CD4 lymphocyte counts ≥300cells/μL and plasma HIV RNA levels <10,000 copies/ml. They presented no significance differences in estradiol and progesterone levels between both groups. This study was limited by differences in sample size, higher CD4 cell counts and lower HIV RNA levels in the Weinberg versus the WIHS studies, indicating that HIV disease status may influence sex steroid levels. (15) In our study, 66.7% cases in group A, 14.3% in group B, 14.8% in group C and 4.2% in controls had progesterone <0.21ng/dl. (Table 1, Fig 3) Data related to progesterone is scarce and controversial. LIMITATIONS: This cross sectional study was limited by small sample size, use of total testosterone due to constraint of resources which is less useful than free testosterone. Follicle Stimulating Hormone, Luteinizing 73 Hormone and SHBG measurement could have added in understanding the mechanisms behind hormonal perturbations.

Conclusion:
Endocrine involvement in HIV infection starts at an early stage and its severity amplifies with decrease in CD4+ cell counts. If these deficiencies are detected, suspected and treated in HIV management, quality of life in HIV positive patients can be improved significantly.  Declaration of conflicting interest: None  Funding: None  Author contributions: Dr. Asha Kumari and Dr. Shashi Seth conceived the idea of study, planned the study and collected the samples. Dr. Piyush helped in statistical evaluation of data. Dr. Uma Choudhary and Dr. Veena Singh Ghalaut helped in conducting the study and Dr. Pawan helped in conducting patient's examination.  Acknowledgements: Arun Kumar for motivation