CHLAMYDIAL INFECTION, PLASMA PEROXIDATION AND OBESITY IN TUBAL INFERTILITY

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A.C. Nsonwu-Anyanwu¹, M.A.Charles-Davies¹, A.A.Oni², V.O. Taiwo³ and F.A. Bello⁴

1. Department of Chemical Pathology, University College Hospital, Ibadan.
2. Department of Medical Microbiology & Parasitology, University College Hospital, Ibadan.
3. Department of Veterinary Pathology, University College Hospital, Ibadan.
4. Department of Obstetrics and Gynaecology, University of Ibadan, Nigeria.

Abstract

Background: Genital tract infections and obesity are both sources of oxidative stress. Alterations in immune and antioxidant parameters may arise from this or from an indeterminate autoimmune mechanism.

Objective: This study aimed to investigate the association of Chlamydial infection, obesity and oxidative response with tubal infertility in Nigerian women.

Methods: It was a case-control study of 40 women with tubal infertility and 32 fertile women, respectively, recruited from the Infertility and Family Planning Clinics respectively, of the University College Hospital, Ibadan, Nigeria. Anthropometric indices were measured in each subject and endocervical swabs were taken to screen for current genital tract infection. Antioxidant, hormonal and immunologic analysis were performed on serum.

Results: None of the subjects had current genital tract infections. Chlamydia trachomatis IgG positivity was significantly higher in infertile than in fertile subjects [OR 4.33; 95%CI (0.078-0.681)]. No significant variations were observed in the anthropometric indices, antioxidant parameters and hormones between infertile and the fertile women. Body mass index correlated positively with oxidative stress in infertile subjects. Waist and hip circumferences correlated negatively with oestradiol in women with tubal infertility.

Conclusion: Chlamydial infection is associated with tubal factor infertility, however, obesity seems to increase oxidative stress and reduce fertility potential in women with tubal factor infertility.

Keywords: Tubal infertility, obesity, oxidative stress, Chlamydia

Introduction

Human reproductive failure is as old as mankind. This public health problem involves all regions of the world and its prevalence worldwide varies from 10-15%¹; 10-32% in Africa² and 31.5% in Nigeria³. The various categories of human reproductive failure include: infertility, recurrent pregnancy loss (spontaneous miscarriages or abortions and preterm birth) and ectopic pregnancy. Causes include environmental and lifestyle factors, congenital malformations, endocrine disorders, immunologic abnormalities and sequelae of genital tract infections^4,5.

The association of infertility with genital tract infections (GTIs) or sexually transmitted infections has been demonstrated. GTIs associated with human reproductive failure include: Treponema pallidum, Neisseria gonorrheae, Chlamydia trachomatis, Trichomonas vaginalis and Schistosoma haematobium infections among others. The spread of gonococcal and Chlamydial infection to upper female genital tract may cause pelvic inflammatory disease with severe tubal scarring leading to tubal infertility⁶. Genital tract infections evoke both cellular and humoral immune response leading to activation of polymorphonuclear leucocytes, macrophages and the release of cytokines. These immunologic factors produced in response to GTIs influence various aspects of reproduction including follicle development, ovulation, luteinisation, oocyte quality, fertilization, implantation, foetal development and pregnancy immunotolerance^7-9.

Abnormal immunologic profile which in part is characterized by autoantibody abnormalities have been described in infertile women¹⁰. GTIs induce increased production of reactive oxygen species and nitric oxide leading to oxidative stress which has been implicated in the pathophysiology of tubal and peritoneal factor infertility, endometriosis, preeclampsia, hydatidiform mole, free radical-induced birth defects and abortions^11-16. Their pathological effects are exerted by various mechanisms including lipid peroxidation, peroxidative DNA damage, mitochondrial alterations and apoptosis¹⁷. The complex interplay between GTI and its induction of oxidative stress and pathologic immune response both systemic and locally and the deleterious effects of these responses on the male and female genital tract which include tissue damage, tubal scarring, pelvic inflammatory disease, chronic salpingitis, endometritis and distal tubal obstruction have been implicated as pathologic mechanisms of infertility.

Excessive weight and central distribution of body fat have been related to an increased risk of normogonadotrophic anovulation¹⁸. High body mass index (BMI) has been implicated in development of insulin resistance and hyperinsulinaemia. Elevated insulin levels have been reported to inhibit the conversion of testosterone to oestrogen. Lower levels of oestrogen will lead to anovulation and infertility¹⁹. Obesity appears to have a major impact on reproductive performance and can compromise it in a variety of ways; including menstrual disorders and anovulation. Weight reduction in obese patients reduces hyperandrogenism and hyperinsulinaemia; both of which factors influence the ovarian response to follicular stimulating hormone. Weight loss can reestablish ovulation in obese anovulatory patients or improve their response to ovulation induction. Clark et al. ²⁰ in 1995 found that weight loss re-established ovulation in obese anovulatory patients or improved their response to ovulation induction: in a series of 67 anovulatory women, 90% resumed ovulation after weight loss and 78% conceived. The same study group confirmed these findings in a larger series²¹. Similar results were obtained in preliminary observations by Crosignani et al., 1999 ²² and 2002 ²³. In another series, among the 27 out of 33 patients with irregular menstrual cycles who lost weight, ¹⁸ re-established regular cycles ¹⁸. A total of 60% had ovulatory levels of plasma progesterone after weight loss. Neither menstrual cycle improvement, nor ovulatory values of progesterone nor pregnancies occurred in the eight patients who did not lose weight. Therefore, it is presumed that obesity may be responsible for the relative ovarian insensitivity to infertility treatment¹⁸.

This study aimed to investigate the association of Chlamydial infection, obesity and oxidative response with tubal infertility in Nigerian women.

MATERIALS AND METHODS
Study design
This case control study was conducted in the University College Hospital (UCH), Ibadan, Nigeria. It served as a pilot for an on-going larger work studying the association of oxidative stress and pathologic response to stress with infertility. The study population comprised of female patients of reproductive age attending the Infertility Clinic and a control group of age-matched fertile women who were new clients at the Family Planning Clinic. The study protocol was approved by UI/UCH Ethical Committee (Ref UI/EC/08/0083).

Hypothesis
The hypothesis was that tubal infertility is not associated with oxidative stress and obesity. The outcome measures were the anthropometric and oxidative stress parameters of the subjects.

Selection of Subjects
Forty consenting women with infertility of at least one year’s duration (with tubal blockage identified by hysterosalpingography) were recruited from the infertility clinic, while 32 controls were recruited from the family planning clinic. The controls were women without previous infertility who had childbirth within the last two years, and who had not been on any form of contraception prior to recruitment. Exclusion criteria included women that were undergoing any form of contraceptive therapy, previous history of uterine surgery, malignancy, long term medication, chronic organ or systemic illness and those who did not give consent.

Anthropometric indices (height, weight, waist and hip circumferences) were taken to calculate the body mass index and waist-hip ratio (WHR) respectively.

Sample Collection
Ten millilitres of venous blood samples were collected aseptically from each subject on days 3-5 and 21-23 of a 28-30 day menstrual cycle, respectively. The samples were dispensed into universal containers. After clot retraction, the samples were centrifuged at 3000 rev/s for ten minutes after which serum was extracted and stored in small aliquots at -20oC. High vaginal swabs (HVS) and endocervical swabs (ECS) were also taken from all subjects of study using sterile swab sticks for detection of common sexually-transmitted infections.

Laboratory Methods
Endocrinological analysis: follicular stimulating hormone (FSH), luteinizing hormone (LH), progesterone (P4), estradiol (E2) and prolactin (PRL) were measured with enzyme immunoassay method (EIA) from Immunometrics Ltd., London, UK.

Antioxidant profile: total antioxidant potential (TAP) was estimated using the ferric reducing antioxidant power (FRAP) method of Benzie and Strain²⁴. Total plasma peroxides (TPP), as a biomarker of Oxidative Stress (OS), were estimated using the modified FOX 2 method of Harma et al.²⁵. Oxidative stress index (OSI) was calculated as a ratio of TPP/TAP.

Microbiological Analysis: screening for Trichomonas vaginalis was by microscopy while Neisseria gonorrheae was by gram staining, followed by culture if necessary. Immunologic analysis: endocervical swabs were tested for Chlamydia trachomatis antigen with a rapid screening test (DiaSpot™, Bresta Perkasa, Indonesia). Serum was screened for Treponema pallidum antibodies (IgG & IgM) by immunochromatographic method with Exact® syphilis diagnostic device, USA; and Chlamydia trachomatis antibodies (CT IgG) with ImmunoComb®, Orgenics Ltd., Yavne, Israel.

Statistical Analysis
Data was analyzed using the statistical package of social science (SPSS) software 15.0 version. For quantitative variables, paired student’s t-test was used to test for mean differences. Pearson’s correlation analysis was employed to determine associations between variables. For non-quantitative variables,  -square analysis was used for determination of associations between variables. Significant p was <0.05.