Routine Screening and Treatment of Asymptomatic Abnormal Vaginal Flora Before ART: Focus on Clindamycin and Clotrimazole

Introduction to ART

Assisted reproductive technologies (ART) are defined as an act of handling or manipulating the oocyte in vitro prior to replacement, either as an oocyte or an embryo.¹Cheung LP. Patient selection for assisted reproductive technology treatments. Hong Kong Med J. 2000 Jun;6(2):177-83.   Additionally, the 1992 Fertility Clinic Success Rate and Certification Act described ART as the procedure that engage the handling of both sperms and eggs. Due to the rapid advancements in the area of ART, infertile couples may seek direct referral for this technique, despite trying simpler measures including ovulation induction and intrauterine insemination. The prevalence of infertility in couples living in developed country is reported to be 15%. However, since past decades there has been no major change in the prevalence of infertility, but there has been an increase in demand for treatment, due to the availability of ART treatments. The common ART treatments comprise IVF, gamete intrafallopian transfer, intracytoplasmic sperm injection, microsurgical epididymal sperm aspiration, testicular sperm extraction, and frozen embryos (Table 1).¹Cheung LP. Patient selection for assisted reproductive technology treatments. Hong Kong Med J. 2000 Jun;6(2):177-83.  ²Myers ER, McCrory DC, Mills AA, et al. Effectiveness of Assisted Reproductive Technology. Rockville (MD): Agency for Healthcare Research and Quality (US); 2008 May. (Evidence Reports/Technology Assessments, No. 167.) 1, Introduction. Available from:http://www.ncbi.nlm.nih.gov/books/NBK38554/  

Initially, ART was used only for patients with tubal infertility. However, presently it is being used for almost all types of infertility cases, since its success rate is expected to be higher in comparison to other conventional treatments. Common indications for ART include previous fertilization failure, unexplained infertility, moderate-tosevere endometriosis, male-factor infertility, and failure of other treatments. Despite its several uses in different indications, ART treatment also has disadvantages. Its use impose more expenses over the patients, and has potentially serious complications including ovarian hyperstimulation syndrome and increased chances of multiple pregnancies.The treatment of infertility must therefore be personalized to avoid subjecting patients to unnecessary risks due to overtreatment, such as the needless use of ART treatments,or to inappropriate delay due to undertreatment (Table 2). ¹Cheung LP. Patient selection for assisted reproductive technology treatments. Hong Kong Med J. 2000 Jun;6(2):177-83.  

Vaginosis: A Complication Affecting ART

Altered vaginal microbiota has been demonstrated to affect parturition. It has been established that known pathogens such as Mycoplasma tuberculosis, Chlamydia trachomatis, and Neisseria gonorrhoeae can cause infertility and subclinical changes in the microbiota in states like bacterial vaginosis (BV), are also thought to be risk factors in subfertility. Normally, the urogenital site consists of 9% of the total human microbiome, the structure and function of

which demonstrate that the vaginal site has low diversity,signifying that it has the least diverse micropopulations with a preponderance of Lactobacillus species. These species promote a healthy and supportive environment in the preand periconceptual period, not only by their presence but also by the production of lactic acid, hydrogen peroxide (H2O2), bacteriocins, antibiotic toxic hydroxyl radicals,and probiotics. The production of lactic acids and other compounds make the environment of vagina unfavorable habitat for a variety of microorganisms including BV.³Sirota I, Zarek SM, Segars JH. Potential Influence of the Microbiome on Infertility and Assisted Reproductive Technology. Seminars in reproductive medicine. 2014;32(1):35-42.  

Bacterial vaginosis is defined as a disturbance in the Lactobacillus-dominated environment of the vagina or an overgrowth of Gardnerella vaginalis. It is associated with infertility, PTB, endometritis, pelvic inflammatory disease,and increased risk of acquiring human immunodeficiency virus. Additionally, in ART treatments, BV is reported to be a significant cause of miscarriages, preterm delivery and maternal infectious morbidity.3 A study4 was conducted to evaluate the effects of BV on the rates of conception and miscarriage in females undergoing IVF. The study enrolled 867 consecutive women undergoing IVF, who were screened for BV with a Gram stained vaginal smear before egg collection. Results divulged that 24.6% of women were found to have BV. Among women with BV,31.6% had a significantly increased risk of miscarriage in the first trimester in comparison to 18.5% with normal vaginal flora.⁴Ralph SG, Rutherford AJ, Wilson JD. Influence of bacterial vaginosis on conception and miscarriage in the first trimester: cohort study.BMJ. 1999 Jul 24;319(7204):220-3.   Similarly, in a recent meta-analysis⁵Leitich H, Kiss H. Asymptomatic bacterial vaginosis and intermediate flora as risk factors for adverse pregnancy outcome. Best Pract Res Clin Obstet Gynaecol. 2007 Jun;21(3):375-90.   BV was found to be a risk factor for preterm delivery and maternal infectious morbidity and a strong risk factor for late miscarriage. The meta-analysis included 14 new studies with results for 10,286 patients, so that results for 30,518 patients in 32 studies were available. Results revealed that BV doubled the risk of preterm delivery in asymptomatic patients and in patients with symptoms of preterm labor. It also considerably increased the risk of late miscarriages and maternal infection in asymptomatic patients.⁵Leitich H, Kiss H. Asymptomatic bacterial vaginosis and intermediate flora as risk factors for adverse pregnancy outcome. Best Pract Res Clin Obstet Gynaecol. 2007 Jun;21(3):375-90.  

Importance of Routine Screening and Treatment of Asymptomatic Vaginal Flora Before ART

Increased risk of complications due to altered vaginal microbiome, has compelled the physicians to go for routine screening and treatment (if required) of asymptomatic vaginal flora prior to ART. The clinical treatment protocols for infertility with in vitro fertilization pre-embryo transfer (IVF-ET) offer a unique opportunity to evaluate the human vaginal microbiome in defined, shifting hormonal milieu and study the potential associations of the vaginal microbiome community with cycle outcome of pregnancy. There exists evidence in terms of published data, demonstrating the reduced pregnancy rates in women undergoing IVF-ET with concurrent vaginal infections with cultured bacteria. This propels the need for early detection and treatment of the condition.⁶Hyman RW, Herndon CN, Jiang H, et al. The dynamics of fertilization-embryo transfer. Journal of Assisted Reproduction and Genetics. 2012;29(2):105-115.  

Role of Clindamycin and Clotrimazole in the Treatment of Abnormal Vaginal Flora

Majority of females experience at least one episode of vaginitis during their lifetime and some of them also have recurrent infections.⁷Irving G, Miller D, Robinson A, Reynolds S, Copas AJ. Psychological factors associated with recurrent vaginal candidiasis: a preliminary study. Sex Transm Inf 1998;74:334-338.   There exist a variety of pathogenic agents in vaginal microflora, which are responsible for a variety of infections in females. The presenting symptoms of these infections may appear to be similar, but are caused by different micro-organisms. Abnormal vaginal discharge,itching, burning sensation, irritation and discomfort are frequent complaints among females attending obstetrics and gynecology clinics. Nonetheless, several vaginal infections present with few or no symptoms. Thus, there is a need to adopt different therapeutic strategies. ⁸Mulu W, Yimer M, Zenebe Y, Abera B. Common causes of vaginal infections and antibiotic susceptibility of aerobic bacterial isolates in women of reproductive age attending at Felegehiwot referral Hospital,Ethiopia: a cross sectional study. BMC Women’s Health. 2015;15:42.  

Accurate treatment modality includes appropriate diagnosis of the condition by the use of routine staining method, culture and sensitivity reports and molecular probes. Nevertheless, the sensitivity of the routine method is uncertain, since it is not 100% accurate. Besides, if molecular probes are used which are the most accurate method for diagnosis; it will impose a huge economical burden and need of expertise to run them will be troubling. Mixed infection is very common involving Candida albicans or Gardnerella vaginalis, which can be dealt with oral therapies or combination of local treatment.⁹Daswani BR, Sambarey PW, Ghongane BB, Bhardwaj R, Naik S, Palewar M. A Comparative Open Labeled Clinical Study to Evaluate the Efficacy and Tolerability of Two Different Intravaginal Formulations Containing a Combination of Clindamycin and Clotrimazole in Patients of Bacterial, Trichomonal, Candidial and Mixed Vaginal Infections. Available at: www.ctri.nic.in/Clinicaltrials/pmaindet2.php?trialid=2922.   The empiric treatment of co-infections is becoming a routine,as cost of treatment is less expensive than lab testing for the organisms. The most common chemotherapeutic agents used in the treatment of vaginitis are antibacterial agents like metronidazole, tinidazole; antibiotics like amoxicillin and clindamycin; and antifungal agents like clotrimazole,miconazole (used either orally or vaginally).¹⁰Menard J-P. Antibacterial treatment of bacterial vaginosis: current and emerging therapies. International Journal of Women’s Health. 2011;3:295-305.   However,the treatment success rates as low as 52% and recurrence rates 50% within 6-12 months are commonly reported.⁹Daswani BR, Sambarey PW, Ghongane BB, Bhardwaj R, Naik S, Palewar M. A Comparative Open Labeled Clinical Study to Evaluate the Efficacy and Tolerability of Two Different Intravaginal Formulations Containing a Combination of Clindamycin and Clotrimazole in Patients of Bacterial, Trichomonal, Candidial and Mixed Vaginal Infections. Available at: www.ctri.nic.in/Clinicaltrials/pmaindet2.php?trialid=2922.  ¹¹Murphy, Kathleen. “Probiotics for recurrent bacterial vaginosis.” Australian Journal of Medical Herbalism 22.3 (2010): 104+. Academic One File. Web. 23 Sept. 2016.   Hence, a need for treatment that covers all the common pathogens associated with vaginal infections is mandatory.

As previously mentioned, majority of vaginosis are mixed infections and symptoms tend to overlap, which make it hard to differentiate between bacterial and fungal infection. When treated with a single agent alone symptoms may persist, revisits to doctor will add to patient inconvenience and cost. Furthermore, secondary infection such as vaginal candidosis frequently occurs within the month following treatment with an antibiotic.¹⁰Menard J-P. Antibacterial treatment of bacterial vaginosis: current and emerging therapies. International Journal of Women’s Health.2011;3:295-305.   The use of antibiotic-antifungal combination usually ensures coverage of a broad range of organisms and polymicrobial infections. This reality became the stimulus for the initiation of combination antibiotic therapy using an agent with excellent anaerobic bacterial coverage together with an agent with good antifungal properties.¹²Kozinn PJ, Taschdjian CL. Combined Antifungal and Antibiotic Therapy. British Medical Journal. 1967;3(5560):307-308.  

Combination regimes with clindamycin have become a standard for treatment of these infections. A number of combinations of antibiotic and antifungal agents (both local and systemic) are available, which further substantiate the benefits of this combination treatment. Clindamycin and clotrimazole combination brands are already available in market. Clindamycin and clotrimazole are widely used,well known and do not cause major side effects when used locally.⁹Daswani BR, Sambarey PW, Ghongane BB, Bhardwaj R, Naik S, Palewar M. A Comparative Open Labeled Clinical Study to Evaluate the Efficacy and Tolerability of Two Different Intravaginal Formulations Containing a Combination of Clindamycin and Clotrimazole in Patients of Bacterial, Trichomonal, Candidial and Mixed Vaginal Infections. Available at: www.ctri.nic.in/Clinicaltrials/pmaindet2.php?trialid=2922.  ¹⁰Menard J-P. Antibacterial treatment of bacterial vaginosis: current and emerging therapies. International Journal of Women’s Health. 2011;3:295-305.  

Clotrimazole

Clotrimazole (an imidazole derivative) is a broad spectrum anti-fungal agent that brings about its therapeutic effect by the alteration of fungal cell membrane permeability.It inhibits the synthesis of ergosterol which alters the permeability of the cell membrane of sensitive fungi and leads to structural and functional impairment of the cytoplasmic membrane. The cell membrane then becomes unable to function as a selective barrier, resulting in loss of potassium and other cellular constituents (Figure 1). It has its fungicidal action against Candida albicans, other species of Candida and also has trichomonacidal action. ⁹Daswani BR, Sambarey PW, Ghongane BB, Bhardwaj R, Naik S, Palewar M. A Comparative Open Labeled Clinical Study to Evaluate the Efficacy and Tolerability of Two Different Intravaginal Formulations Containing a Combination of Clindamycin and Clotrimazole in Patients of Bacterial, Trichomonal, Candidial and Mixed Vaginal Infections. Available at: www.ctri.nic.in/Clinicaltrials/pmaindet2.php?trialid=2922.  ¹³National Center for Biotechnology Information. PubChem Compound Database; CID=2812, https://pubchem.ncbi.nlm.nih.gov/compound/2812 (accessed Sept. 23, 2016).  

Clindamycin

Clindamycin on the other hand is a semi synthetic derivative of lincomycin, primarily acting against gram positive and wide range of anaerobic bacteria. It inhibits bacterial protein synthesis by its action at bacterial ribosome. Clindamycin preferentially binds to the 50S ribosomal subunits and affects the process of protein synthesis (Figure 2). It has a potent antimicrobial activity against Bacteroides spp., Gardnerella vaginalis, Mobiluncus spp.,Mycoplasma hominis, Peptostreptococcus spp., Haemophilus spp.and Corynebacterium.⁹Daswani BR, Sambarey PW, Ghongane BB, Bhardwaj R, Naik S, Palewar M. A Comparative Open Labeled Clinical Study to Evaluate the Efficacy and Tolerability of Two Different Intravaginal Formulations Containing a Combination of Clindamycin and Clotrimazole in Patients of Bacterial, Trichomonal, Candidial and Mixed Vaginal Infections. Available at: www.ctri.nic.in/Clinicaltrials/pmaindet2.php?trialid=2922.  ¹⁴Seli E, Arici A, Barbieri RL, et al. Ovulation induction with Clomifene citrate. Available at: http://cursoenarm.net/UPTODATE/contents/ mobipreview.htm?22/32/23041?source=HISTORY [Accessed on 22/9/2016].  

Evidence Based Efficacy of Clindamycin and Clotrimazole in Treating Abnormal Vaginal Flora

Clindamycin is an effective agent for the treatment of abnormal vaginal flora related to vaginosis. A study¹⁵Hashim HA. Management of women with clomifene citrate resistant polycystic ovary syndrome – an evidence based approach. Available at: http://cdn.intechopen.com/pdfs-wm/29510.pdf [Accessed on:22/9/2016].   was conducted to determine its efficacy in reducing the risk of preterm birth and late miscarriage in women with abnormal vaginal flora at ¹⁵Hashim HA. Management of women with clomifene citrate resistant polycystic ovary syndrome – an evidence based approach. Available at: http://cdn.intechopen.com/pdfs-wm/29510.pdf [Accessed on:22/9/2016].   Similar results were shown by another study¹⁶ESHRE Capri Workshop Group. Health and fertility in World Health Organization group 2 anovulatory women. Hum Reprod Update.2012;18(5):586-99.   which demonstrated that treatment of asymptomatic abnormal vaginal flora and bacterial vaginosis with oral clindamycin early in the second trimester considerably reduced the rate of late miscarriage and spontaneous preterm birth in a general obstetric population.¹⁶ESHRE Capri Workshop Group. Health and fertility in World Health Organization group 2 anovulatory women. Hum Reprod Update.2012;18(5):586-99.

Since, the combination regime has become a treatment of choice for mixed infections; the addition of clotrimazole to clindamycin is also proved to be beneficial. Based on the above mentioned evidences, a strong case can be made for the administration of combined use of clotrimazole and clindamycin in patients with asymptomatic abnormal vaginal flora before ART.

Conclusion

Assisted reproductive technologies are defined as an act of handling or manipulating the oocyte in vitro prior to replacement, either as an oocyte or an embryo. Altered vaginal microflora manifested as vaginosis is a serious complication of ART. It leads to pre-term delivery,miscarriages, and maternal infectious morbidity in patients undergoing ART therapy. Majority of vaginosis are mixed infections and symptoms tend to overlap, which make it hard to differentiate between bacterial and fungal infection. Thus, to overcome this, a combination regime of clindamycin and clotrimazole is considered as the standard treatment of these infections.

References

1. Cheung LP. Patient selection for assisted reproductive technology treatments. Hong Kong Med J. 2000 Jun;6(2):177-83.
2. Myers ER, McCrory DC, Mills AA, et al. Effectiveness of Assisted Reproductive Technology. Rockville (MD): Agency for Healthcare Research and Quality (US); 2008 May. (Evidence Reports/Technology Assessments, No. 167.) 1, Introduction. Available from:http://www.ncbi.nlm.nih.gov/books/NBK38554/
3. Sirota I, Zarek SM, Segars JH. Potential Influence of the Microbiome on Infertility and Assisted Reproductive Technology. Seminars in reproductive medicine. 2014;32(1):35-42.
4. Ralph SG, Rutherford AJ, Wilson JD. Influence of bacterial vaginosis on conception and miscarriage in the first trimester: cohort study.BMJ. 1999 Jul 24;319(7204):220-3.
5. Leitich H, Kiss H. Asymptomatic bacterial vaginosis and intermediate flora as risk factors for adverse pregnancy outcome. Best Pract Res Clin Obstet Gynaecol. 2007 Jun;21(3):375-90.
6. Hyman RW, Herndon CN, Jiang H, et al. The dynamics of the vaginal microbiome during infertility therapy with in vitro fertilization-embryo transfer. Journal of Assisted Reproduction and Genetics. 2012;29(2):105-115.
7. Irving G, Miller D, Robinson A, Reynolds S, Copas AJ. Psychological factors associated with recurrent vaginal candidiasis: a preliminary study. Sex Transm Inf 1998;74:334-338.
8. Mulu W, Yimer M, Zenebe Y, Abera B. Common causes of vaginal infections and antibiotic susceptibility of aerobic bacterial isolates in women of reproductive age attending at Felegehiwot referral Hospital,Ethiopia: a cross sectional study. BMC Women’s Health. 2015;15:42.
9. Daswani BR, Sambarey PW, Ghongane BB, Bhardwaj R, Naik

S, Palewar M. A Comparative Open Labeled Clinical Study to Evaluate the Efficacy and Tolerability of Two Different Intravaginal Formulations Containing a Combination of Clindamycin and Clotrimazole in Patients of Bacterial, Trichomonal, Candidial and Mixed Vaginal Infections. Available at: www.ctri.nic.in/Clinicaltrials/pmaindet2.php?trialid=2922.

10. Menard J-P. Antibacterial treatment of bacterial vaginosis: current and emerging therapies. International Journal of Women’s Health. 2011;3:295-305.
11. Murphy, Kathleen. “Probiotics for recurrent bacterial vaginosis.”Australian Journal of Medical Herbalism 22.3 (2010): 104+. Academic One File. Web. 23 Sept. 2016.
12. Kozinn PJ, Taschdjian CL. Combined Antifungal and Antibiotic Therapy. British Medical Journal. 1967;3(5560):307-308.
13. National Center for Biotechnology Information. PubChem Compound Database; CID=2812, https://pubchem.ncbi.nlm.nih.gov/compound/2812 (accessed Sept. 23, 2016).