1 Department of Obstetrics and Gynecology and 2 Laboratory for Microbiology, Ha'Emek Medical Center, Afula and 3 Rappaport Faculty of Medicine, TechnionIsrael Institute of Technology, Haifa, Israel
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Abstract |
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Key words: antibiotic susceptibility/assisted reproduction technology/bacterial colonization/embryo transfer/uterine cervix
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Introduction |
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In this study we assessed whether the nature of bacterial flora, found in the uterine cervical canal at embryo transfer, affects the rate of conception in ART cycles.
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Materials and methods |
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The protocols for embryo transfer of frozenthawed embryos were two: (i) sonographic and biochemical (serum LH and progesterone) detection of spontaneous ovulation; (ii) endometrial estrogenic build-up, commencing at day 2 of menstruation and consisting of 48 mg/day oral micronized estradiol until endometrial thickness reached 9 mm, when 50 mg/day progeterone in oil was added. The diagnosis of conception was made on the basis of positive serum HCG testing and demonstration of a gestational sac in the uterine cavity. Patients were monitored for signs and symptoms of genital infection during the study and up to 2 weeks after embryo transfer.
When the patient was prepared for the embryo transfer procedure, the vaginal portion of the cervix was cleaned with dry gauze, a sham catheter was introduced into the cervical canal and the distal 10 mm piece was cut into a tube containing 1 ml of sterile saline solution for sampling. Embryo transfer was performed immediately after this. Within 1 h after sampling, the contents of the tube with the 10 mm catheter piece were shaken vigorously. Ten µl aliquots of this suspension were seeded onto the following plates: two plates of blood agar (Geloise du Sang, Sanofi Pasteur, Marnes-La-Coquetter, France, supplemented with 5% human blood), one plate of MacConkey agar (Oxoid, Basingstoke, UK), chocolate agar (Eugonagar; Becton and Dickinson, USA, supplemented with chocolatized human blood, haemin and nicotinamide adenine dinucleotide), potato dextrose agar (Difco, Detroit, USA), Centre for Disease Control (CDC) anaerobic agar (Geloise du Sang) and modified SP4 agar for mycoplasma/ureaplasma (Clarke, 1992). C.trachomatis was not sought, because the prevalence of positive cultures in asymptomatic women in Israel is very low (Ghinsberg and Nitzan, 1994
). The rest of the fluid was centrifuged and the bottom 50 µl was seeded on the same plates as above. All plates were incubated for 48 h aerobically at 37°C, but the CDC anaerobic agar plates and one of the plain blood agar were incubated in an anaerobic jar for 72 h and then examined.
All bacterial growth was quantified as follows: every single colony growing in the 10 µl inoculated plates represented 100 colony forming units (CFU)/cm in the original 1 cm tip. Any bacteria growing in the centrifuged inoculation, but not in the first inoculation, were included in the category `<100 CFU/cm'. Samples showing more than three different organisms in similar counts were defined as `mixed culture'.
Bacterial isolates were identified by Gram stain, conventional biochemical methods, latex agglutination and automatic identification by Microscan system (Dade, Behrung, West Sacramento, USA). The adequate routine method was performed in each case.
Antibiotic susceptibility testing for aerobic bacteria was performed by disk diffusion test (Kirby and Bauer method) according to the standards of the National Committee for Clinical Laboratory Standards (NCCLS) (M2-A6, 1999). Antibiotic susceptibility testing for anaerobic bacteria was performed by E-test method (AB-Biodisk, Sweden) as recommended by NCCLS guidelines (M2-A6, 1999).
Statistical analysis
Statistical analysis was conducted with the t-test, MannWhitney U-test, 2 and regression analysis as applicable. No power calculation was done because the study was observational.
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Results |
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The specific susceptibility to antibiotics among the group of pathogens, as observed in the 129 positive cases, reveals that all were resistant to cefazoline. All Gram-positive and 90% of the Gram-negative bacteria were sensitive to amoxicillin/clavulanic acid compound.
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Discussion |
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Bacterial colonization of the uterine cervix has been suspected to influence conception rate. Possible causes of this could be an association between a cervical-positive culture and a concomitant, pre-existing uterine infection (Templeton and Morris, 1998); or direct inoculation of the endometrium or the embryo as a result of passage through the colonized cervix (Czernobilsky, 1978
; Paulson et al., 1990
; Tabibzadeh and Babaknia, 1995
). The settlement of this issue is beyond the scope of clinical studies. The above notwithstanding, the incomplete and differing results of the existing studies do not provide enough ground for concrete recommendations on the preferred mode of intervention in this regard, which would be expected to improve conception rates (Fanchin et al., 1998
). In order to collect data which will enable a firm conclusion and recommendation, we examined not only the type of bacteria present in the uterine cervix, but also quantified it and performed detailed antibiotic susceptibility tests. Although there seems to be a relationship between the colony count and chances for conception, there could not be a defined cut-off count, beyond which it would be preferable to cancel embryo transfer. We also added a `natural' control group in the form of cycles in which frozenthawed embryos were transferred. This in turn allowed evaluation of the effect of our oocyte retrieval and the routine antibiotic preventive treatment on cervical findings. Interestingly, the colonization in women both after recent oocyte retrieval and in those scheduled for embryo transfer of frozenthawed embryos was not susceptible to cefazoline, the drug that we used for prophylaxis during oocyte retrieval. This may explain the absence of difference in colonisation between the fresh and the frozen cycle patients.
Whereas our study results delineate a significant association between cervical colonization and the chance for conception, they suggest but do not provide statistically significant evidence for an association with either a bacterial type or the number of CFUs. Thus, our findings of E.coli in 8.5% of the positive cultures do not agree with another published study (Fanchin et al., 1998) that described a significant predominance of E.coli (68%) among the positive cultures, implying an association with conception rates. It has been suggested (Egbase et al., 1996
) that there is a correlation between the number of colonies and the type of bacteria and the chance for conception. In this regard, it should be pointed out, that unlike ours, both these studies examined only aerobic bacterial colonization. An additional aspect of the results of these two studies and this one is the differing bacterial colonization profiles among different patient populations. This may indicate different future interventions for these respective populations.
Regarding the true nature of the association that we found, it is unclear whether there is a causative role for bacterial colonization in decreasing conception, or whether these two phenomena are merely the result of an obscure common factor. Nevertheless, our findings on the profile of antibiotic susceptibility pave the way for an interventional study, which will examine this issue in our patient population.
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Notes |
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Submitted on November 24, 2000; resubmitted on May 4, 2001
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References |
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accepted on October 15, 2001.