Article Does intravaginal probiotic supplementation increase the pregnancy rate in IVF embryo transfer cycles?

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1 RBMOnline - Vol 11. No Reproductive BioMedicine Online; on web 27 April 2005 Article Does intravaginal probiotic supplementation increase the pregnancy rate in IVF embryo transfer cycles? Dr Yinon Gilboa was born in Jerusalem in 1966 and concluded his medical studies, cum laude, at the Technion, Israel Institute of Technology in He then started his residency at the Obstetric and Gynecology Department of Rabin Medical Centre. The study presented here was conducted during his IVF training. His present fields of interest are obstetrics and obstetric ultrasound. Dr Yinon Gilboa Yinon Gilboa, Itai Bar-Hava, Benjamin Fisch, Jacob Ashkenazi, Isachar Voliovitch, Tamar Borkowski, Raoul Orvieto 1 Department of Obstetrics and Gynecology, Rabin Medical Centre, Petah Tiqva, and Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel 1 Correspondence: orvieto@clalit.org.il Abstract This study investigated the effect of intravaginal administration of probiotics immediately after oocyte retrieval on vaginal colonization and outcome of the IVF embryo transfer cycle. One hundred and seventeen women who underwent ovarian stimulation and IVF were randomized immediately after oocyte retrieval into two groups: those who received intravaginal probiotics (study group, n = 50) and those who did not (control group, n = 67). Vaginal colonization with lactobacilli and pregnancy rate were compared between the two groups. No significant between-group differences were observed in patient age, oestrogen and progesterone concentrations on day of human chorionic gonadotrophin administration, number of oocytes retrieved, fertilization rate, number of embryos transferred, or pregnancy rate. The presence of lactobacilli in the vagina during oocyte retrieval or embryo transfer did not improve the pregnancy rate. Furthermore, intravaginal administration of lactobacilli following oocyte retrieval did not affect the prevalence of lactobacilli during embryo transfer, or the pregnancy rate. Intravaginal probiotic supplementation immediately after oocyte retrieval has no effect on vaginal colonization or pregnancy rate in IVF cycles. Keywords: colonization, IVF, lactobacilli, pregnancy rate Introduction The presence of a pelvic infection during IVF apparently has no effect on the number of oocytes recovered, fertilized and cleaved. However, it does pose a risk for conception if it appears at the critical time of implantation (Orvieto and Ben- Rafael, 2001). In one study, patients with mechanical infertility had a 23 31% pregnancy rate after IVF embryo transfer, whereas those with pelvic inflammatory disease (PID) had zero pregnancies (Ashkenazi et al., 1994). Lactobacilli are part of the normal vaginal flora in most women. Lactobacilli generate and release H 2 O 2, which leads to the inhibition and destruction of adjacent organisms, particularly in the presence of peroxidase (Klebanoff et al., 1996). Moreover, the decrease or absence of lactobacilli from the vaginal flora is associated with the overgrowth of pathogenic organisms such as Gardnerella vaginalis and certain anaerobes, leading to bacterial vaginosis (Klebanoff et al., 1991). This finding raises the possibility that probiotic therapy could restore the normal vaginal flora and improve pregnancy rates. Probiotic therapy involves the administration of live microorganisms in amounts that confer a health benefit on the host (FAO/WHO, 2001). The underlying mechanism of action of these supplements includes the production of pathogen-inhibitory substances, replenishment of beneficial bacteria, and reduction of the action of microbial toxins. Studies of bacteria isolated from the vagina have shown that the presence of lactobacilli is associated with an increased 71

2 72 pregnancy rate (Moore et al., 2000; Salim et al., 2002). Furthermore, oral or vaginally administered Lactobacillus strains can safely colonize the vaginal ecosystem and displace and kill pathogens such as Gardnerella vaginalis and Escherichia coli, thereby modulating the immune response to interfere with the inflammatory cascade that leads to implantation failure (Chlebecek and Reich, 1993; Reid et al., 1995, 2001, 2003; Jeavons, 2003). In patients undergoing assisted reproduction technologies, the effect of vaginal or cervical colonization with bacteria on IVF embryo transfer cycle outcome is still controversial. Some studies reported a favourable effect of cervico vaginal colonization with lactobacilli (Moore et al., 2000; Salim et al., 2002) and a detrimental effect with other pathogenic microorganisms (Witkin et al., 1994, 1995; Egbase et al., 1996, 1999; Fanchin et al., 1998; Moore et al., 2000; Salim et al., 2002), whereas others showed no effect of lactobacilli (Liversedge et al., 1999; Ralph et al., 1999). The detrimental effect of cervico vaginal colonization with pathogenic bacteria might be explained by the presence of a concomitant, pre-existing uterine infection (Templeton and Morris, 1998). It is also possible that the passage of the bacteria through the colonized cervix, with direct inoculation of the endometrium or embryo, interferes with implantation (Czernobilski, 1978; Paulson et al., 1990; Tabibzadeh and Babaknia, 1995). The aim of the present study was to further evaluate the association between vaginal colonization with lactobacilli and pregnancy rate during IVF embryo transfer cycles, and to determine if intravaginal administration of probiotics immediately after oocyte retrieval restores vaginal lactobacilli colonization and improves IVF embryo transfer cycle outcome. Materials and methods Protocol The study sample consisted of infertile patients attending the IVF unit who met the following criteria: age <38 years, normal uterine cavity, and no hydrosalpinges. Patients who had a chronic illness or were receiving chronic medical treatment and patients who had repeated IVF failures (>3 previous cycles) were excluded. Each patient was thoroughly counselled and gave informed consent before participating in the study. The study was approved by the Hospital Committee for Ethics in Human Research. Patients were excluded from the analysis if no oocytes were retrieved or embryo transfer was not undertaken. Ovarian stimulation consisted of either a mid-luteal long gonadotrophin releasing hormone analogue (GnRHa) suppressive protocol or the short GnRHa flare protocol. When two or more follicles of at least 18 mm in mean diameter were present and the hormonal profile was satisfactory, 10,000 IU of human chorionic gonadotrophin (HCG) was administered. Oocytes were retrieved transvaginally h later, under ultrasound guidance. Before oocyte retrieval, swabs were rubbed on the vaginal wall and transferred for culture or were rolled on a glass slide for Gram staining. The vagina was then washed extensively with normal saline and oocyte retrieval was performed. Thereafter, some patients received 2 capsules of intravaginal probiotics (Probiotic Femina; Altman Ltd, Holon, Israel) (study group) and others did not (control group). Each capsule of Probiotic Femina contains 3 billion live cells of human strains of Lactobacillus acidophilus, Bifidobacterium bifidum and Bifidobacterium longum. The groups were allocated by pseudo-randomization according to the week of oocyte retrieval. Routine IVF intracytoplasmic sperm injection (ICSI) was then performed as appropriate. The techniques for sperm processing and oocyte and embryo handling have been detailed elsewhere (Bar-Hava et al., 1997; Krissi et al., 2004). Transvaginal embryo transfer was performed h after oocyte retrieval. Swabs for culture and Gram staining were obtained again from the vagina at embryo transfer. Transfer was completed after the vagina and cervix were washed with gamete culture medium and the cervical mucus was removed. All patients received luteal support with natural progesterone. Clinical pregnancy was defined as visualization of a gestational sac by ultrasound and elevation of serum HCG concentrations. Statistical analysis Statistical analysis was performed using Student s t-test and Fisher s exact test, as appropriate. Results are presented as means ± SD. P < 0.05 was considered statistically significant. According to calculations, in order to detect an improvement in pregnancy rate from 30 to 50%, the observed pregnancy rate in the current programme and the estimate pregnancy rate after probiotic administration, with a type 1 error of 0.05 and 80% power, 103 patients were needed in each study group. Results One hundred and seventeen women were included in the study, 67 in the control group and 50 in the study. The demographic data and ovarian stimulation protocols of the participants are shown in Table 1. There were no between-group differences in causes of infertility or type of ovarian stimulation protocol. Moreover, no statistically significant differences were found between the groups in patient age, oestradiol and progesterone concentrations on the day of HCG administration, number of oocytes retrieved, fertilization rate, number of embryos transferred, or pregnancy rate. The prevalence of lactobacilli and the corresponding pregnancy rates are presented in Table 2. There was no difference in the prevalence of lactobacilli before oocyte retrieval between the study and control groups (38 and 58.2% respectively), and there was no association of the presence of lactobacilli before oocyte retrieval or embryo transfer and pregnancy rate. Surprisingly, within the control group, patients in whom lactobacilli were present in the vagina during both oocyte retrieval and embryo transfer showed a tendency toward a lower pregnancy rate than those without lactobacilli (29.1 and 50% respectively, P < 0.08). Intravaginal probiotic supplementation immediately after oocyte retrieval had no effect on vaginal colonization by lactobacilli. Analysis of the control and study patients who had

3 Table 1. Patients demographic data and ovarian stimulation protocols. Control group (no Study group intravaginal (intravaginal probiotic; n = 67) probiotic; n = 50) Age (years) 30.2 ± ± 4.1 Oestradiol concentration on day of 4030 ± ± 1969 HCG administration (pmol/l) Progesterone concentration on day 3.3 ± ± 1.2 of HCG administration (nmol/l) No. oocytes retrieved 9.75 ± ± 6.2 Fertilization rate 64 ± ± 22 No. embryos transferred 2.48 ± ± Pregnancy rate (%) There were no significant differences between groups. Table 2. Prevalence of lactobacilli and corresponding pregnancy rates in the two groups. Before oocyte retrieval Before embryo transfer Lacto- No. Prevalence Pregnancy Lacto- No. Prevalence Pregnancy bacillus patients (%) rate (%) bacillus patients (%) rate (%) Whole sample (n = 117) Probiotic group (n = 50) Control group a (n = 67) a a P < 0.05 between the groups. 73

4 74 no lactobacilli before oocyte retrieval showed that lactobacilli reappeared before embryo transfer in 28.5% of the control patients and 35.4% of the study patients. Discussion Pelvic inflammatory disease appearing at the critical time of implantation in the IVF cycle may adversely affect the chance of pregnancy (Orvieto and Ben-Rafael, 2001). Some researchers claim that the introduction of endotoxin-releasing bacteria (even at subclinical concentrations) into the peritoneal cavity during transvaginal oocyte recovery, and into the uterine cavity or tubes during embryo transfer, can cause oocyte degeneration and fragmentation (Ng et al., 1987). The local pelvic inflammatory reaction may also be involved in the adverse effect on pregnancy. Bacteria trigger a chain of events that lead to the activation, proliferation and differentiation of lymphocytes and the production of specific antibodies and various cytokines. Cytokines play a direct role in implantation and early embryonic development, and their excess production at this stage may disrupt the delicate balance between the immune and reproductive systems and result in reproductive failure (Ben-Rafael and Orvieto, 1992). Furthermore, circulating cytokines such as interleukin (IL)-1 and tumour necrosis factor (TNF), which act on the central nervous system, may also indirectly affect the outcome of IVF embryo transfer by inducing a temperature elevation. Studies have suggested that temperature elevation is essential for amplifying the emergence of T-cell immunity in peripheral tissues (Hanson, 1993), and that it leads to disintegration of the cytoskeleton (Kitano and Okada, 1990), and may affect the transport of organelles. Whether vaginal colonization, and specifically the presence of lactobacilli, a part of the normal vaginal flora, exerts an effect on the chance of pregnancy in IVF cycles is controversial. Egbase et al. (1996) reported a correlation among the number of colonies, type of bacteria retrieved from the tip of the embryo transfer catheter, and the chance for conception. Thereafter, they demonstrated (Egbase et al., 1999) that the routine administration of prophylactic antibiotics at the time of oocyte retrieval was associated with a reduction in positive microbiology cultures of embryo catheter tips 48 h later. Moreover, implantation and clinical pregnancy rates were significantly lower in women with positive microbial cathetertip cultures. Salim et al. (2002) examined whether the nature of the bacterial flora found in the uterine cervical canal at embryo transfer is associated with the rate of conception in assisted reproduction. They found that 23 of the 75 patients (30.7%) with sterile or Lactobacillus-positive cervical cultures conceived, compared with only 21 of the 129 patients (16.3%) in whom a pathogenic microorganism was recovered. Any Gram-negative colonization was associated with failure of conception. The authors concluded that failure to conceive in assisted reproduction is significantly associated with bacterial colonization of the uterine cervix, but they did not delineate either a bacterial type or number. By contrast, Fanchin et al. (1998) noted a significant predominance of E. coli (68%) among the positive cultures, implying an association between microbial flora of the cervix and poor IVF embryo transfer outcome. Therefore, lactobacilli, which are able to colonize the vaginal and cervical mucosa and inhibit pathogen binding and growth, would be expected to serve as effective probiotic factors for the prevention of vaginal infection. In the present study, however, no effect of the presence of lactobacilli in the vagina during oocyte retrieval or embryo transfer could be demonstrated on pregnancy rate. This might be explained by the similar distribution of aerobes and anaerobes in pregnant and infertile patients, as observed by Moberg et al. (1978). The findings are in accordance with the study of Liversedge et al. (1999), which found that bacterial vaginosis did not have an adverse effect on fertilization rate or early pregnancy outcome. There was no significant difference in implantation rates among women with bacterial vaginosis (15.8%), and intermediate flora (13.1%) or normal flora (15.5%). The fertilization rates were significantly higher in the bacterial vaginosis group, but this may have been due to its significantly greater proportion of women with tubal infertility, which is associated with more favourable fertilization rates than other types of infertility. With regard to probiotics specifically designed for women s health, the administration of lactobacilli by mouth or intravaginally has been shown safely to reduce the risk of urinary tract infection, bacterial vaginosis, and yeast vaginitis (Reid et al., 1995, 2001, 2003) by preventing the growth and adhesion of various intestinal and urogenital pathogens. However, in the present study, their intravaginal administration following oocyte retrieval did not enhance pregnancy rate. Therefore, while the presence of lactobacilli may affect the vaginal flora, it probably has no influence on uterine colonization. Furthermore, since vaginal washing prior to oocyte retrieval and embryo transfer is efficient for preventing PID (Orvieto and Ben Rafael, 2001), it is possible that it might also prevent direct microbial inoculation of the endometrium or embryo. In the present study, the intravaginal administration of 6 billion lactobacilli following oocyte retrieval also had no effect on the prevalence of lactobacilli during embryo transfer. The observation places the potential role of intravaginal probiotics in the IVF embryo transfer cycle in doubt. The aim of this study was not to provide a detailed inventory of the cervical flora, but rather to investigate whether the presence of lactobacilli in the cervix with or without prior intravaginal probiotic administration affects IVF outcome. Further studies are needed to assess the effects of different durations, types and routes of probiotic administration following oocyte retrieval on all microbial flora. References Ashkenazi J, Farhi J, Dicker D et al Acute pelvic inflammatory disease after oocyte retrieval: adverse effects on the results of implantation. 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