|Year : 2018 | Volume
| Issue : 1 | Page : 7-13
The effect of bacterial colonization of the embryo transfer catheter on Outcome of In vitro Fertilization–Embryo transfer treatment
Roy N Maduka1, James A Osaikhuwuomwan2, Michael E Aziken2
1 Department of Obstetrics and Gynaecology, College of Medical Sciences, University of Benin, Benin City; Department of Obstetrics and Gynaecology, General Hospital, Agbor, Delta State, Nigeria
2 Department of Obstetrics and Gynaecology, College of Medical Sciences, University of Benin, Benin City, Nigeria
|Date of Web Publication||2-Jul-2018|
James A Osaikhuwuomwan
Department of Obstetrics and Gynaecology, College of Medical Sciences, University of Benin, Benin City
Source of Support: None, Conflict of Interest: None
Background: In vitro fertilization–embryo transfer (IVF − ET) has become a core treatment method for managing infertility. Bacterial contamination of the ET catheter may affect outcome, but there is still no consensus of evidence. Objectives: This study aims to assess the effect of bacterial colonization of the ET catheter tip on the clinical pregnancy rate in an IVF–ET treatment. Methods: An analytical cross-sectional study among women undergoing IVF–ET treatment was undertaken. The patients selected had both cervical swab and the tip of the ET catheter cultured. The patients were grouped into positive (bacterial isolated) and negative (no bacterial isolated) based on the culture result. The clinical pregnancy rate (primary outcome) between the two groups was compared. Results: A total of 80 patients were selected. In 34 patients (42.25%), the cervical culture was positive, while 46 patients (57.50%) had negative cervical culture. Catheter tip culture was positive in 27 patients (33.75%) and negative in 53 patients (66.25%). The predominant microorganisms isolated were Escherichia coli (23.75%), Staphylococcus spp. (18.75%), and Streptococcus spp. (15.00%). The clinical pregnancy rate was 26.25%. The significant factors affecting clinical pregnancy were the age of the patient (P = 0.044), duration of infertility (P = 0.01), and culture result (P = 0.03). Conclusion: Bacterial colonization of the ET catheter tip is associated with a reduction in the clinical pregnancy rate. Utility of routine cervical swab; microscopy, culture, and sensitivity at recruitment of patients for IVF–ET treatment is highlighted.
Keywords: Bacterial colonization, clinical pregnancy, embryo transfer, infertility, in vitro fertilization
|How to cite this article:|
Maduka RN, Osaikhuwuomwan JA, Aziken ME. The effect of bacterial colonization of the embryo transfer catheter on Outcome of In vitro Fertilization–Embryo transfer treatment. Afr J Med Health Sci 2018;17:7-13
|How to cite this URL:|
Maduka RN, Osaikhuwuomwan JA, Aziken ME. The effect of bacterial colonization of the embryo transfer catheter on Outcome of In vitro Fertilization–Embryo transfer treatment. Afr J Med Health Sci [serial online] 2018 [cited 2018 Nov 20];17:7-13. Available from: http://www.ajmhs.org/text.asp?2018/17/1/7/235739
| Introduction|| |
In vitro fertilization and embryo transfer (IVF–ET) is increasingly being utilized for the management of infertility as a result of the continued development of newer techniques and improving results of treatment with IVF–ET. Approximately 50% of infertile couples will require treatment with some form of assisted reproduction technology in other to achieve pregnancy. Despite this progress, the majority of IVF cycles still do not produce a viable pregnancy.
The outcome of IVF is affected by several factors including patients and clinical characteristics., These include variables such as the age of the patient, duration of infertility, the stimulation protocol used, number of embryos transferred, and ET technique among others. Notwithstanding the considerable progress in the assisted reproduction field, the implantation rate of replaced embryos remains low.,, Embryo implantation has been described as the main event that limits the success of IVF–ET. Factors that might interfere with the normal embryo implantation process include the patients' age, endometrial receptivity, embryo quality, and transfer technique as well as microorganism infection of the genital tract.,,, As IVF treatment involves the placement of embryos into the uterine cavity using a catheter that passes through the cervix, the possibility of bacterial contamination during the ET procedure clearly exists.
The role of microbial infection on the outcome of IVF–ET treatment has been studied by several authors with contradictory result. Genital infections, particularly those caused by sexually transmitted microorganisms, Neisseria More Details gonorrhoea and Chlamydia trachomatis, may be found in the endocervix. These are some normal bacteria flora may contaminate ET catheter as it passes through the endocervix. The detection of Chlamydia species in the endocervix of women undergoing IVF–ET has been associated with decreased implantation rates and ongoing pregnancy rates., There is also growing evidence that bacterial contamination of the uterine cavity can occur during embryo replacement through the tip of the ET.,,,, A case of severe pelvic infection complicating transcervical ET in an embryo recipient (with no risk of from transvaginal oocyte retrieval) has been reported. This report suggested that clinical infection can occur from passage of the transfer catheter transcervically into the uterine cavity.
However, the localization of such microorganisms requires the use of both aggressive swabs to collect endocervical tissue or catheter tip samples and expensive diagnostic procedures that hinder its routine application., In women undergoing IVF treatment, research has shown that participants who test negative for bacterial contamination have approximately 50% higher pregnancy rates than those who test positive for bacterial contamination resulting from ET catheter tip.
There are evidence gaps in the literature on the effect of bacteria colonization of the ET catheter on clinical pregnancy. The studies available on the topic are few, and there is no consensus of opinion. More so, there are very limited research data in our own environment (Sub-Saharan Africa) as IVF–ET is relatively new. Additional research is therefore needed to assess the association of bacteria colonization of the ET catheter and IVF–ET outcome.
This study is designed to evaluate the effect of bacterial contamination (endocervical microbial isolate) of the catheter tip on the clinical pregnancy rate in an IVF–ET treatment at the University of Benin Teaching Hospital.
| Methods|| |
This study was analytical, cross-sectional study.
This study was conducted at the Human Reproductive Research Programme (HRRP) Unit of the Obstetrics and Gynaecology Department of the University of Benin Teaching Hospital, Benin. The hospital serves as a major referral center in the Niger Delta region of Nigeria. HRRP is a dedicated infertility treatment unit with assisted reproduction techniques services in a public tertiary Hospital. The IVF–ET treatment is usually done in batches of 20–30 couples following the synchronization of their cycle combined oral contraceptives. Patients batched for IVF between the months of January to July 2014 at the HRRP center of UBTH formed the study population. All consecutive women undergoing IVF–ET at the HRRP who met the inclusion criteria during the study period were recruited as participants. Informed consent was obtained, and catheter tip microscopy, culture, and sensitivity were done at the microbiology laboratory of the HRRP Unit.
All consecutive patients undergoing IVF–ET treatment at the University of Benin Teaching hospital who do not have any of the exclusion criteria were included.
Exclusion criteria included
Patients who used donated eggs, patients with uterine abnormality, confirmed cases of endometriosis, and patients with significant cardiovascular, pulmonary, neurological, allergic, hepatic, or renal diseases. Each patient participated only once in the study.
Approval for this study was obtained from the Ethics and Research Committee of the University of Benin Teaching Hospital. This study was carefully explained to the participants and their informed consent obtained before being recruited into the study. Participants were assured of anonymity and confidentiality, the rights of patients to participate or not were respected.
The IVF–ET treatment protocol at the HRRP involved the standard practice of pituitary downregulation, followed by ovarian stimulation with gonadotropin using the long protocol during which follicular response was assessed by regular transvaginal ultrasound. Human chorionic gonadotropin (hCG) was administered for ovulation induction when follicles of at least 18 mm diameter were obtained and oocyte retrieved by the transvaginal route performed 34–36 h after the hCG administration. Intravenous co-amoxiclav 1.2 g was given prophylactically to all patients at oocyte retrieval. In a previous study by Brook et al., where co-amoxiclav was administered prophylactically to patients undergoing IVF–ET treatment, there was 50.6% reduction in catheter contamination. For ethical reasons, co-amoxiclav was administered to all patients in this study in line with the unit protocol. Those patients who still had positive bacteria growth after co-amoxiclav administration were regarded as the culture positive group. Following in vitro treatment, the resulting embryo(s) were transferred on the 3rd day transcervically using a sterile standard ET catheter (HG Wallace Ltd UK). ET was done aided with abdominal ultrasound. Posttransfer luteal support with progesterone.
Specimen collection and processing
The specimen for this study was the tip of the HG Wallace catheter used for ET during the IVF–ET treatment. A sterile Cusco's speculum was inserted into the vagina to expose the cervix. First, a sterile swab stick was used to collect specimen from the cervical os. All embryos were transferred using the sterile flexible Wallace catheter type, and a nontouch sterile replacement technique was used. Contact between the transfer catheter and the vaginal wall was avoided. Following each successful ET, the embryologist cutoff the distal 2 cm of the transfer catheter using a sterile scissors. The catheter tip was placed in a sterile container and along with the cervical swab stick specimen was sent immediately to the microbiology laboratory for analysis and culture. The result of catheter tip and cervical culture was retrieved after 48–72 h. The patients as routinely practiced did their pregnancy test 2 weeks after ET. For the patients with positive pregnancy test, an ultrasound scan was done on the 8th week to confirm clinical pregnancy. For the purpose of this study, follow-up was ended with the detection or otherwise of clinical pregnancy at the 8th week ultrasound assessment.
In the laboratory, specific analysis carried out included the following:
- Microscopy – Smears of the cut catheter tip and cervical swab were made on sterile glass slide, and Gram staining technique was used to detect the presence of bacteria
- Culture – The catheter tip and cervical swab stick were used to inoculate into various media, which included blood agar, chocolate agar, MacConkey agar, and Thayer–Martins medium. These were incubated for 48 h, following which the different media were examined for bacteria growth
- Biochemical test – Media plates with bacteria growth (positive plates) were further subjected to various biochemical tests to identify the bacteria isolate, using standard procedure and algorithm.
The laboratory results were stated as positive or negative growth, and where the culture is positive, the bacteria isolate were stated accordingly.
This study was limited to culture of aerobic organisms. Anaerobes were not cultured, as we do not have the facilities for anaerobic culture. Samples were not assessed for Chlamydia and Mycoplasma hominis because our specimen collection method (catheter tip test) could not meet the adequate technical conditions required for screening for these bacteria (the presence of epithelial cells). Furthermore, sensitivity pattern of cultured microbes was not analyzed.
Designed pro forma was used to record important demographic information and clinical correlates for each patient including age, parity, cause of infertility, duration of infertility, baseline follicle-stimulating hormone (FSH) level, endometrial thickness, result of catheter culture, number of embryo transferred, clinical pregnancy, and implantation rate. The generated database was analyzed using the SPSS version 16 (Chicago, SPSS Inc). Categorical variables were expressed as absolute numbers and percentages and analyzed using the Chi-square test or Fisher exact test where appropriate, whereas continuous variables were presented as means with standard deviations and the differences were analyzed with the t-test. A multivariate analysis was used for the data analysis to control for cofounding variables where appropriate. The level of significance was set as P < 0.05
| Results|| |
This study was conducted between January and July 2014. They were a total of 116 patients for IVF--T during the study period. Thirty-six patients were excluded from the study; among the excluded patients, 32 of them did not meet the inclusion criteria, while the catheter was inadvertently discarded by the embryologist in 4 of the patients. Out of the 80 patients, 34 (42.55%) had positive cervical culture, while 46 (57.50%) had negative cervical culture. There was positive microbial growth in the catheter tip in 27 patients (33.75%), while 53 patients (66.25%) had no bacteria growth on the catheter tip. None of the patients with negative cervical growth had positive catheter tip culture. On the other hand, seven patients (8.75%) had negative catheter tip culture even though cervical culture was positive.
When the patients were grouped into 2 based on the results of catheter tip culture results (positive or negative), [Table 1], there was no significant difference between the groups with regard to the sociodemographic and clinical characteristics of the patients (mean age 32.04 ± 4.60 vs. 33.04 ± 3.68, P = 0.30; mean parity 0.15 ± 0.36 vs. 0.23 ± 0.51, P = 0.47; mean duration of infertility 7.44 ± 2.75 vs. 7.20 ± 2.17, P = 0.63; Baseline FSH 8.58 ± 7.20 vs. 9.90 ± 6.50, P = 0.44; and endometrial thickness 9.70 ± 1.91 vs. 10.27 ± 2.52, P = 0.31).
|Table 1: Sociodemographic and clinical characteristics of the study group|
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The different microorganisms isolated from the catheter tips are as shown on [Table 2]. Organisms isolated from the catheter tip were not different from those isolated from the endocervical swab culture in all cases. The organisms identified were Escherichia More Details coli 19 (23.75%), Staphylococcus spp. 15 (18.75%) Streptococcus spp. 12 (15.00%), Lactobacillus 6 (7.50%), Proteus spp. 6 (7.50%), Klebsiella 3 (3.75%), and Pseudomonas 2 (2.5%). A considerable number of patients tested positive to more than one microorganism.
Analysis of the different variables affecting IVF–ET treatment (clinical pregnancy) is shown on [Table 3]. Pregnancy test was positive in 25 patients (pregnancy rate of 31.25%) but with 4 being biochemical pregnancies. Overall, there were 21 (26.25%) confirmed clinical pregnancies while in the remaining 59 (73.85%), IVF–ET treatment failed.
There was significant difference between the groups with positive clinical pregnancy and those with negative clinical pregnancy in terms of the mean age of patients (31.19 ± 3.95 vs. 33.24 ± 3.94, P = 0.04), duration of infertility before the current IVF–ET treatment (6.84 ± 2.36 vs. 8.03 ± 2.25, P = 0.009), and whether catheter tip culture was positive or negative for microbial growth (P = 0.033).
Multivariate logistic regression of the significant variables affecting clinical pregnancy [Table 4] showed that the three variables were still significant. Age of the patient (odds ratio [OR] 0.864, 95% confidence interval [CI] 0.747–1.000), duration of infertility (OR 0.679, 95% CI 0.504–0.914), and catheter tip culture (OR 7.357. 95% CI 1.597–33.88) significantly influenced clinical pregnancy rate. Subanalysis showed implantation rate of 14.02% (23/126; number of ultrasound demonstrated gestational sac divided by total number of embryo transferred). The variables statistically significant for implantation were the duration of infertility, (P = 0.007) and whether the catheter tip culture was positive or negative (P = 0.001). The patients age (P = 0.07), baseline FSH (P = 0.28), and endometrial thickness (P = 0.80) were not significant statistically. There were 5 cases (6.25%) of multiple pregnancies. Two (2.5%) cases were in the positive culture group, while 3 (3.75%) were in the negative culture group. There was no statistical difference between the two groups.
| Discussion|| |
Although few reports on bacterial contamination at ET and its implication in infertility treatment have been published, evidence implicating bacterial infection as detrimental to IVF outcomes is steadily increasing. In this study, bacterial contamination of the tip of the ET catheter had a negative effect on IVF–ET treatment outcome. Our study did not detect any difference between the groups with regard to clinical characteristics, namely, indication for IVF–ET, baseline FSH, endometrial thickness, number of embryo transferred, and the ease of ET. However, the age of the patients, the duration of infertility, and whether catheter tip culture was positive or negative for microbial growth were factors significantly associated with clinical pregnancy. These characteristics of the study group, therefore, allow us to speculate that the presence of microorganisms on the tip of the ET catheter may influence the clinical pregnancy rate.
Overall, the rate of catheter tip contamination in this study (33.75%) was lower compared to previous reports in literature., The use of co-amoxiclav prophylactically before ET and our inability to culture anaerobic microorganisms in this study would have contributed to a lower catheter tip bacterial contamination in this study. The most common microorganism cultured was E. coli (23.75%) which was closely followed by Staphylococcus and Streptococcus species. This is similar to previous findings in this subregion where bacterial colonization of the lower genital tract among women of childbearing age was studied. The study by Fanchin et al. found a higher preponderance of E. coli (68%). Similarly, the study by Franchin et al. was limited in its finding due to the authors' inability to culture anaerobes.
Clinical pregnancy rate following IVF treatment varies from center to center. In a 2 years review by Ikechebelu et al. working in a private IVF center in Southeast Nigeria, the clinical pregnancy rate was 30%. While in a comprehensive 5 years review by Orhue et al. in this center, the clinical pregnancy rate was 30%. Similarly, in this study, the clinical pregnancy rate was 26.25%.
The significant factors that affected clinical pregnancy rate were the age of the patients, the duration of infertility, and whether catheter tip culture was positive. The relationship between age and duration of infertility is not unexpected as they are known negative predictors of success in IVF–ET treatment., There is, however, no consensus on the association between the presence of bacteria on the catheter tip and outcome of IVF–ET. While bacterial contamination of the ET catheter was a significant variable that affected clinical pregnancy in this study, other studies were inconclusive.,,,
In support of our study findings, Fanchin et al. had conducted a prospective qualitative analysis of catheter tips bacteria colonization of women undergoing IVF–ET treatment. They observed clinical pregnancy rate was 24% in the culture-positive group and 37% in the culture-negative group, while implantation rate was also higher in the culture-negative group compare to the culture-positive group. The conclusion was that the presence of microbial flora of the cervix on the ET catheter tip is associated with poor IVF–ET outcome. Other studies have also demonstrated that the presence of vaginal–cervical microbial contamination at the time of ET is associated with significantly decreased pregnancy rates.,
Several pathophysiological mechanisms have been advanced to explain the decrease in clinical pregnancy following bacterial contamination of the ET catheter. Bacterial contamination may decrease the embryos capacity to implant due to the effects on the embryo itself and/or the endometrium., Before implantation, the zona pellucida of the embryo which has a barrier function against infection is lost at the cleavage stage, thus exposing the embryo to the detrimental effect of bacteria. It has also been proposed that any acute inflammatory response in the endometrium will generate cytokines, macrophages, prostaglandins, and leukotrienes resulting in some form of subclinical endometritis with consequent deleterious effect on embryo implantation., Although all patients in this study had co-amoxiclav prophylaxis, the growth of microorganism on the catheter tip and cervix of 33.75% and 42.55%, respectively, is still high. The finding may suggest the need to review the antibiotic sensitivity pattern of the common microorganisms encountered in our environment. Like in our study, Egbase et al. could not culture for anaerobic organism but instead administered metronidazole routinely at the point of ET. They recommended a prospective study to determine whether eradication of endocervical microorganisms is possible and whether their eradication will improve implantation rates. In the study by Ismail  concluded that although antibiotics significantly reduced catheter tip contamination rate, there was no difference detected in the clinical pregnancy/IVF–ET outcome. Their finding may be explained by the small size of the population sample. Embryo implantation rate has been reported to decrease with increasing age of women., While age did not significantly affect implantation rate in this study, bacterial contamination of catheter tip and duration of infertility significantly influenced implantation.
| Conclusion|| |
In conclusion, this study has demonstrated that contamination of the ET catheter tip during ET in IVF–ET treatment exists. More importantly, bacterial contamination of the catheter tip was associated with reduction in the clinical pregnancy rate. In view of the high rate of catheter tip contamination despite the routine use of co-amoxiclav as prophylaxis for preventing infection during egg retrieval; the need to determine the prevalence, type, and sensitivity pattern of microbial colonization of our healthy female populace is important as this may influence prophylaxis for patients undergoing IVF–ET in the future. We recognize significant study limitations such as inability to culture anaerobes or document antibiotic sensitivity pattern of the cultured microbes; a small population size as well as multiple confounders.
The practice of IVF–ET is still evolving in Sub-Saharan Africa, and the need to develop strategies that will improve outcome cannot be overemphasized. We can recommend utility of routine cervical swab; microscopy, culture, and sensitivity at recruitment of patients for IVF–ET treatment as a tool to improving outcome. Furthermore, the need for further research on the sensitivity pattern of cultured microbes in our environment is suggested.
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Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3], [Table 4]