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Human Reproduction Vol.17, No.2 pp. 337–340, 2002
Bacterial colonization of the uterine cervix and success rate
in assisted reproduction: results of a prospective survey
Raed Salim1, Izhar Ben-Shlomo1,3,4, Raul Colodner2, Yoram Keness and Eliezer Shalev1,3
1Department
of Obstetrics and Gynecology and 2Laboratory for Microbiology, Ha’Emek Medical Center, Afula and 3Rappaport
Faculty of Medicine, Technion–Israel Institute of Technology, Haifa, Israel
4To
whom correspondence should be addressed at: Department of Obstetrics and Gynecology, Ha’Emek Medical Centre, Afula
18101, Israel. E-mail: [email protected]
BACKGROUND: Overgrowth of bacteria in the birth canal is associated with an increased risk of late miscarriage,
preterm labour, post-partum endometritis and low birthweight. Conception rates in assisted reproduction treatments
(ART) remain frustratingly low. We examined whether the nature of bacterial flora, found in the uterine cervical
canal at embryo transfer, is associated with the rate of conception in ART. METHODS: We sampled for
bacteriological culture the cervical canal of 204 patients who underwent embryo transfer. Of these, 139 (68%) were
of fresh embryos, following recent vaginal oocyte retrieval and prophylactic antibiotic therapy, and 65 (32%) of
frozen–thawed embryos, without any vaginal intervention in the preceding days. Bacteriological work-up included
identification, colony count and antibiotic susceptibility profile. Conception was correlated with bacterial type and
colony count. RESULTS: In 75 patients (36.8%) sterile cervical cultures or lactobacillus were recorded. Of these
75 patients, 23 (30.7%) conceived, whereas among the 129 in whom any pathogenic micro-organism was recovered
only 21 (16.3%) conceived (P ⍧ 0.002). No difference in colonization was found between women who underwent
frozen–thawed versus fresh embryo transfer (57 and 67% respectively). Any Gram-negative colonization was
associated with no conception. All Gram-positive, and 90% of the Gram-negative bacteria, were sensitive to
augmentin. CONCLUSIONS: Failure to conceive in ART is significantly associated with bacterial colonization of
the uterine cervix.
Key words: antibiotic susceptibility/assisted reproduction technology/bacterial colonization/embryo transfer/uterine cervix
Introduction
Bacterial vaginosis is associated with an increased risk of late
miscarriage, preterm labour, post-partum endometritis and low
birthweight (Hay et al., 1994; McCoy et al., 1995; Carey
et al., 2000). Several reports indicate a higher risk for the
pregnancy if bacterial vaginosis is present during early stages
of pregnancy (Kurki et al., 1992; Hay et al., 1994, McGregor
et al., 1995). Assuming a putative role for bacterial overgrowth
in all the above gestational pathologies, one is tempted to
speculate an even earlier effect, i.e. in embryonic implantation.
In this regard, it is of note that assisted reproductive technology
(ART) has been used for almost two decades, but conception
rates consistently run lower than expectations (Fanchin et al.,
1998), especially in the light of the high costs involved. Several
studies have addressed the possible association of bacterial
colonization of the vagina and cervix with success in ART,
but their conclusions seem to contradict each other. Whereas
some studies (Egbase et al., 1996; Fanchin et al., 1998) that
sampled the cervical canal during embryo transfer found an
association between the type of flora and the chances for
conception, another study (Ralph et al., 1999) that sampled
the vagina prior to oocyte retrieval did not.
© European Society of Human Reproduction and Embryology
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.
Materials and methods
From June 1 to October 31, 1999, 204 embryo transfers were
performed on days Sunday through to Thursday, from which samples
could be seeded and processed. Of these transfers, 139 were of fresh
embryos and 65 of frozen–thawed embryos. The protocols used for
controlled ovarian hyperstimulation were as previously described
(Shalev et al., 1995; Ben-Shlomo et al., 1997). HCG (Pregnyl;
Organon, Os, The Netherlands) 10 000 IU i.m. was administered
when at least three follicles reached a mean diameter of 18 mm.
Oocyte retrieval was performed transvaginally under ultrasonographic guidance (6.5 MHz probe, Elcsint 1000; Elscint, Haifa, Israel)
32–38 h later. For prophylaxis against infection, a single i.v. dose of
cefazoline 1 g was administered prior to retrieval. Women allergic to
penicillin (five women in this study) were given i.v. erythromycin
300 mg. Oocytes were either inseminated with partners’ spermatozoa
or were subjected to ICSI, as applicable.
The protocols for embryo transfer of frozen–thawed embryos were
two: (i) sonographic and biochemical (serum LH and progesterone)
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R.Salim et al.
detection of spontaneous ovulation; (ii) endometrial estrogenic
build-up, commencing at day 2 of menstruation and consisting of
4–8 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, Mann–Whitney
U-test, χ2 and regression analysis as applicable. No power calculation
was done because the study was observational.
Results
Overall, in 75 patients either a sterile cervical culture was
recorded or only lactobacilus was recovered. The rate of
potentially pathogenic colonisation (any bacteria other than
lactobacillus) in women who underwent embryo transfer of
frozen–thawed embryos was 57% compared with 67% in
cycles of fresh embryo transfer (not significantly different).
None of the patients reported clinical symptoms related to
overt genital infection, neither was such an infection suggested
338
on examination before embryo transfer. There were 36 clinical
pregnancies in the 139 cycles of fresh embryo transfer (25.9%)
and nine (13.8%) in the 65 frozen–thawed embryo transfer
cycles. Of the 75 patients with either sterile or lactobacillipositive cultures, 23 (30.7%) conceived, whereas among the
129 in whom any other micro-organism was recovered only 21
(16.3%) conceived. This difference was statistically significant
(P ⫽ 0.002). As shown in Table I, these two groups of patients
did not differ in background characteristics. Table II reveals
that the two groups did not differ in parameters of response
to treatment. A further breakdown by fresh versus frozen–
thawed embryo transfer revealed that fresh embryo transfer in
women who had a sterile culture or lactobacillus resulted in a
conception rate of 35.5%, whereas fresh embryo transfer in
women who had positive cultures for other organisms resulted
in a conception rate of 20%. This difference was statistically
significant (P ⫽ 0.045).
The association of micro-organisms, recovered from uterine
cervices, with rates of conception is presented in Table III. It
is of note that mycoplasma was not recovered from any of the
SP4 agar plates.
After calculation of the number of colony forming units
(CFU), which were counted after the seeding as described
above, a preliminary association with conception was found.
Since the presence of any Gram-negative colonisation was
associated with no conceptions at all, these cases were deleted
for the purpose of further calculation. No statistically significant
correlation was found between the number of CFUs and
conception rate. However, out of 41 patients with ⬎10 000
CFUs of any bacterial type other than lactobacillus, only four
(9.7%) conceived, whereas of 89 who had ⬍10 000 colonies,
17 (19.1%) conceived.
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.
Discussion
A host of elements during IVF treatment predict poorer success,
including age, duration and type of infertility, the number of
previous attempts at IVF, quality of the embryos, the number
of oocytes and the number of resulting embryos available for
transfer (Isenberg and D’Amato, 1995; Templeton et al., 1996).
In this prospective study we found that the nature of cervical
colonization is an independent and significant factor in the
determination of success in ART.
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
Bacteria in uterine cervix and ART success rates
Table I. Background characteristics in two groups of women from whom cervical cultures were obtained
before embryo transfer
Age in years (SD)
Gravidity (SD)
Parity (SD)
Infertility factor
Male (%)
Female (%)
Anovulation (%)
Tubal (%)
Endometriosis (%)
Mixed (%)
Unexplained (%)
Infertility duration (years) (SD)
Early follicular FSH IU/l (SD)
Previous treatment cycles (SD)
Normal
(n ⫽ 75)
Colonized
(n ⫽ 129)
Significance
31 (6.1)
1.01 (1.61)
0.39 (0.63)
32 (5.5)
1.05 (1.24)
0.40 (0.58)
NS
NS
NS
43 (57.3)
11 (14.6)
7 (9.3)
3 (4)
1 (1.3)
16 (21.3)
5 (6.6)
5.96 (4.26)
6.76 (5.24)
1.85 (1.0)
61 (47.2)
25 (19.3)
11 (8.5)
13 (10)
1 (0.8)
28 (21.7)
15 (11.6)
6.61 (4.39)
6.50 (4.96)
1.91 (1.0)
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS ⫽ not significant.
Table II. Response to treatment in two groups of women from whom cervical cultures were obtained before
embryo transfer
Gonadotrophin ampoules (SD)
Days of stimulation (SD)
Oocytes retrieved (SD)
Fertilized (SD)
Number of embryos transferred (SD)
Hours to embryo transfer (SD)
Cells/embryo (SD)
Implantation rate (%)
PR/embryo transfer (%)
Normal
(n ⫽ 75)
Colonized
(n ⫽ 129)
Significance
36.9 (13.8)
9.8 (2.0)
15.1 (10.1)
8.4 (5.9)
2.6 (1.2)
61.1 (12.0)
4.9 (1.7)
34/195 (17.3)
23/75 (30.7)
39.2 (17.3)
10.1 (2.3)
13.7 (8.4)
7.7 (5.8)
2.7 (1.3)
63.0 (11.7)
5.1 (1.6)
28/348 (8.0)
21/129 (16.3)
NS
NS
NS
NS
NS
NS
NS
P ⫽ 0.001
P ⫽ 0.002
NS ⫽ not statistically significant; PR ⫽ pregnancy rate.
Table III. The distribution of micro-organism types in cervical cultures
taken from women before embryo transfer during assisted reproduction
treatment
Microorganism
Total positive
Conceptions per
embryo transfer
Rate (%)
Gram-negative
Anaerobic pathogens
Gram-positive
Candida species
Mixed
19a
38
46
18
21
0
4
10
4
3
0
10.5
21.7
22.2
14.0
aTwelve
cases of E.coli.
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 frozen–thawed 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 frozen–thawed 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
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R.Salim et al.
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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Submitted on November 24, 2000; resubmitted on May 4, 2001; accepted on
October 15, 2001