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Original articles

Evaluation of the effect of bacterial colonization in cervical mucus on the outcome of intracytoplasmic sperm injection cycles

Farahat, Mohamed; Nouh, Basel; Rageh, Kamal; Ismael, Mahmoud; Fata, Ahmed; Hablas, Wael; Wafa, Yehia

Author Information
Evidence Based Women's Health Journal: May 2014 - Volume 4 - Issue 2 - p 87-91
doi: 10.1097/01.EBX.0000440890.92323.cc
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Abstract

Introduction

The hostility or receptivity of the cervix to spermatozoa is dependent to a great degree on the condition of the cervical mucus, a subject that deserves more attention in the gynecological literature. It is unfortunate that so much attention has been paid to evaluation of tubal patency, determination of ovulation, and analysis of semen, with relative neglect of the cervical factor. In intracytoplasmic sperm injection (ICSI), embryo transfer (ET) is usually performed 72 h after oocyte retrieval. For a standard ET procedure, transfer is accomplished by transcervical cannulation and injection of embryos into the uterine cavity 1.

Embryo implantation is the main event that limits the success of ICSI-ET. Despite the presence of satisfactory embryo morphological analysis as well as adequate histological and histochemical endometrial characteristics, the overall ICSI-ET pregnancy rates remain poor. This indicates that additional factors might interfere with the normal embryo implantation process. These factors range from culture conditions, hormone stimulation, gamete immaturity, or quality of chromosomal abnormality to the possibility of bacterial contamination of the uterine cavity 2. ICSI-ET procedures involve needle puncture of the vagina for egg retrieval and placement of embryos through the cervix; thus, contamination is possible from vaginal-cervical microorganisms, particularly because vaginal antiseptics are usually not used during egg retrieval or ET to avoid injury of the egg or embryos 3. Prophylactic antibiotics administered to women at the time of oocyte retrieval have been considered to be associated with a reduction in positive microbiology cultures of embryo catheter tips 48 h later in 78.4% of patients 4. The minimal inflammation in response to microorganisms that enter the endometrium from the cervix during ET provides another mechanism that could damage the developing embryo and prevent pregnancy 4.

In terms of the mechanisms that may be responsible for damaging the developing embryo, first, it is conceivable that intense concentrations of microorganisms in the cervix may be associated with subclinical chronic endometritis and therefore poor uterine receptivity. Second, the ET procedure may inoculate cervical microorganisms into the uterine cavity, potentially altering the biochemical or the ultrastructural characteristics of the endometrium required for satisfactory embryo implantation and early development. Third, the possible direct contamination of embryos during transcervical ET may prevent their ability to implant 5.

Participants and methods

The aim of this study was to evaluate the effect of bacterial colonization in cervical mucus on the outcome of ICSI cycles.

  • Study setting: The study was carried out from February 2012 to June 2013; 500 participants with infertility were enrolled in the study in the international Islamic center for population studies and research, Al Azhar University, Cairo, Egypt. All the participants were recruited from the outpatient clinic of the Assisted Reproduction Unit.
  • Research design: A prospective follow-up study design was used to carry out the study.
  • Study participants: The study included 500 consecutive patients from among those attending the unit with a diagnosis of infertility who were assigned for treatment with an intracytoplasmic sperm injection (ICSI). The inclusion criteria were as follows: women who were 36 years old or younger, whose uteri were morphologically normal as confirmed by HSG and U/S to limit additional factors that may affect the results of the study. Moreover, none of the women included in this study had clinical evidence of vaginitis or cervicitis or had concurrent use of antibiotics. Each woman participated only once in the study. Ethical committee approval was obtained and all participants provided oral consent for inclusion in the study.
  • Study maneuver: Assessment of history, and abdominal and pelvic examination were also performed. Routine investigations were carried out for all participants according to the protocols applied in the unit. Downregulation and induction of ovulation were initiated for every patient according to the protocol applied. Follicular monitoring and estimation of endometrial thickness were performed using transvaginal ultrasonography every other day starting 6 days after the initiation of gonadotropins. In the study group, cervical swabs were taken with the first folliculometry and screened for chlamydia and other bacterial growth; antibiotics were administered accordingly for positive cases, and then, after administration of hCG and oocytes retrieval, ET was carried out 48–72 h using a standardized technique with a cook ET catheter. A maximum of four embryos were transferred at a time. No antiseptic solution was used to prepare the cervix for ET, but was only washed with isotonic saline. In the control group, a cervical swab was taken and tested for chlamydia and other bacterial growth, but no other antimicrobials were administered even in positive cases, luteal phase support was provided, and patients were scheduled for B-hCG 2 weeks after ET. The participants were then divided into two subgroups: a pregnant group and a nonpregnant group.
  • Bacteriological investigation methods:
    • PCR: was used for the detection of Chlamydia trachomatis. PCR involved three steps, namely, DNA extraction, amplification, and detection of a specific DNA product.
    • Culture: Swabs were cultured on blood agar, chocolate agar, and Mackonky’s agar. The plates were incubated aerobically and anaerobically in 5% CO2 at 37°C. Bacteria were isolated for identification by standard laboratory procedures.

Results

All data were collected, tabulated, and subjected to statistical analysis. Comparison of the sociodemographic characteristics of the study and the control groups showed no statistically significant differences; their age was around 29 years. The BMI was around 26.5, the mean duration of infertility was, respectively, 5.8 and 6.1 years, and the mean FSH level was 6.6 and 6.3, respectively. The participants were mostly housewives. In terms of the cause of infertility in the two groups, Fig. 1 shows no statistically significant difference between the study and the control group in the cause of infertility; it was mainly the male factor (62–64%).

F1-6
Figure 1:
Relation between the study and the control group in terms of the cause of infertility.

In terms of the characteristics of ICSI cycles, there were no statistically significant differences between the two groups in the number of HMG ampoules administered (37–38 ampoules); the duration of stimulation was around 12.5 days, and endometrial thickness at the time of ET ranged from 10.5 to 10.9 mm. Almost eight oocytes were retrieved; four of these were fertilized and almost two embryos were transferred, as shown in Fig. 2.

F2-6
Figure 2:
Comparison between the study and the control group in the characteristics of the intracytoplasmic sperm injection cycle.

In terms of bacterial growth, Figs 3 and 4 indicate that the organisms isolated varied from staphylococci, Klebsiella, Escherichia coli, lactobacilli, and to a lesser extent anaerobic cocci. However, no statistically significant differences could be observed between the study and the control group.

F3-6
Figure 3:
Relation between cultures of bacterial growth in terms of the pregnancy test in the study group.
F4-6
Figure 4:
Relation between cultures of bacterial growth in terms of the pregnancy test in the control group.

The data for chlamydial growth were also similar in the two groups. As shown in Fig. 5, no statistically significant differences were found between them; there were 17.7% and 14.5% positive cases among the pregnant women in the study and the control group, respectively, which was again not significant.

F5-6
Figure 5:
Relation between the study and the control pregnant cases in terms of chlamydia.

Comparison of various combinations of bacterial and or chlamydial growth did not show any statistically significant differences between the pregnant women in the two groups. The incidence of other bacterial growth (in the study group) was 35.56% compared with 30.1% (in the control group). The incidence of associated chlamydia and other bacterial growth (in the study group) was 8.89% compared with 5.83% (in the control group).

Discussion

Embryo implantation is the main event that limits the success of IVF-ET 5. Genital infections, particularly those caused by sexually transmitted microorganisms, rank among the leading causes of infertility 6. The possibility that genital bacterial contamination may interfere with embryo implantation has been suggested as far back as 1978 7. Clinical studies have shown that bacterial contamination of the ET catheter has a significant negative effect on the clinical pregnancy rates 8–10. In addition, cervical sterility at the time of ART procedures cannot be achieved with the routine use of vaginal antiseptics as there is evidence that these solutions have been shown to have a negative impact on the quality of the oocytes collected and the embryos available for transfer 11. Moreover, there is insufficient evidence on the effects of different antibiotic prophylaxis regimens on ART cycle outcomes 4,12,13. The influence of the cervical mucus present at the time of ET and its bacterial contamination on the outcome of ICSI cycles has been highly debated because of conflicting results in the medical literature. Some authors have reported reduced pregnancy rates, whereas others have not. In addition, most studies supported the use of samples of cervical mucus from catheter tips at the time of ET. C. trachomatis is believed to be one of the major causes of cervical factor infertility as a result of the alterations of the epithelium and mucus composition and the presence of inflammatory cells; therefore, the overall impact of cervical disorders on fertility still needs to be assessed 14. It has been suggested that impaired ovarian function and low ovarian response to ovulation induction are associated with C. trachomatis infection 15. Earlier studies have shown that pathogenesis of the disease was not only induced by the infectious agent but also by immune response to infected tissues 14. Detection of Chlamydia species in the endocervices of women undergoing IVF-ET has been associated with decreased implantation rates 16. The cervix is usually colonized by other potentially pathogenic microorganisms that can be introduced into the uterine cavity or may contaminate embryos during ET 17. The effects of this septic milieu on the outcome of embryo implantation are unclear. Egbase and colleagues, reported poor IVF-ET results in patients with positive microbial growth after the catheters used in ETs were cultured. From a pathophysiologic standpoint, at least three mechanisms may be considered to explain the lower pregnancy rates observed among patients with positive cultures: First, it is conceivable that intense concentrations of microorganisms on the cervix may be associated with subclinical chronic endometritis and, therefore, poor uterine receptivity. Second, the ET procedure may inoculate cervical microorganisms into the uterine cavity, potentially altering the biochemical or ultrastructural characteristics of the endometrium required for satisfactory embryo implantation and early development. Third, the possible direct contamination of embryos during transcervical ET may prevent their ability to implant 18.

This study was carried out in light of this controversy and the need to clearly identify the potential value of bacterial contamination of the cervical mucus and the role of antibiotic in the outcome of ICSI cycles. The vaginal flora contain a large variety of bacterial species, including aerobic and anaerobic organisms, as shown by modern microbiologic methods 19. Moreover, the diversity and kinds of organisms that comprise the vaginal microbial community vary among women 20. As the lower genital tract is naturally inhabited by vaginal flora and pathogenic organisms, operative procedures through or adjacent to this field lead to a moderate to high incidence of infection. Therefore, recommendations for antibiotic prophylaxis have been established in many procedures, including vaginal hysterectomy, abdominal hysterectomy, and cesarean section 21. However, unlike most assisted reproductive techniques, these are major operative procedures that may carry a high morbidity rate from infections. With respect to minor operative procedures related to ART, such as during transvaginal oocyte retrieval and ET, there are no clear recommendations by any society [e.g. American society of Reproductive Medicine (ASRM), European Society for Human Reproduction and Embryology (ESHRE), Middle East Fertility Society (MEFS), Mediterranean Society for Reproductive Medicine (MSRM)] or other evidence-based guidelines (e.g. NICE guidelines). However, these procedures have a high possibility of ascending infection from the lower genital tract to the upper genital tract, especially for those procedures that pass through the endocervical canal into the uterine cavity (e.g. intrauterine insemination and ET). As these procedures have only small areas of tissue trauma, it is questionable whether or not antibiotic prophylaxis, the use of antibiotics for the prevention of infection, for these procedures protects against ascending infection. Therefore, antibiotic prophylaxis might play a role in preventing infection in these procedures, but this is yet to be confirmed officially. In essence, in today’s evidence-based medical environment, any recommendation must be built on two main questions: (a) whether ascending infections occur as a result of the procedure and (b) whether this results in a decreased pregnancy rate in such cases. Only then can a proper set of guidelines be proposed to answer this clinical query.

The present study aimed to evaluate whether the presence of cervical microorganisms in cervical mucus at ET has an effect on the success of pregnancy in women undergoing ICSI procedure, and to determine the benefit of antimicrobials on the outcome of ICSI cycles. The present study has shown that there were no statistically significant differences between the study group and the control group in terms of their age, duration of infertility, and serum FSH level measured on day 3 of the cycle. This was important to enable comparison of pregnant versus nonpregnant women in terms of the presence of chlamydia and other organisms, and to investigate the relation between the presence of these microorganisms and the success or failure rates of the process of ICSI. Similarly, there were also no statistically significant differences between the two groups, the study and the control group, in the number of retrieved oocytes and the number of transferred embryos. This is again to ensure that both groups had equal chances of success and of achieving pregnancy. The present study has shown that there were no statistically significant differences between the two groups, the study and the control group, in bacterial growth in pregnant women, despite administration of antibiotics to the study group according to culture and sensitivity tests. The findings of the present study have also shown that the participants in the study group, who were positive for chlamydia and received anti-chlamydial medications, showed no statistically significant difference from other positive chlamydial cases in the control group who did not receive any anti-chlamydial medications in terms of the pregnancy rate.

Conclusion

The present study reports that chlamydial infection and microbial flora of the cervix detected during ET play no role in the implantation process, and do not affect pregnancy rates significantly in women undergoing the ICSI procedure for the treatment of infertility, and antimicrobials play no significant role in improving the outcome of pregnancy rate.

Acknowledgements

Conflicts of interest

There are no conflicts of interest.

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Keywords:

bacterial colonization; cervical mucus; Chlamydia; intracytoplasmic sperm injection

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