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Relationship of cytopathology and cervical infection to outcome of in-vitro fertilization and embryo transfer
International Journal of Gynecology and Obstetrics (2008) 101, 21–26
a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / i j g o
CLINICAL ARTICLE
Relationship of cytopathology and cervical infection to outcome of in-vitro fertilization and embryo transfer Ying Wang ⁎, Changjuan Wang, Jie Qiao, Lina Wang, Siqun Liang Reproductive Medical Center, Peking University Third Hospital, Beijing, China Received 31 July 2007; received in revised form 17 September 2007; accepted 25 September 2007
KEYWORDS Atypical squamous cells of undetermined significance; In-vitro fertilization and embryo transfer; Squamous intraepithelial lesion; Thin-layer preparation cytologic test
Abstract Objective: To determine whether a relationship exists between in-vitro fertilization and embryo transfer (IVF-ET) outcome and cervical infection or presence of human papillomavirus (HPV). Method: Cervical scrapes, digital colposcopies, and cervical biopsies were performed in 1044 Chinese women undergoing IVF for tubal infertility or, in their partners, abnormal semen. The pregnant (n = 415) and nonpregnant (n = 629) groups differed neither in clinical signs of cervical inflammation nor in rate of HPV detection. Results: There were no associations between IVF-ET outcome and infection rate, degree of cytopathologic abnormality, detection of HPV, or results of digital colposcopy and cervical biopsy. Cytologic results did not correlate with any of the clinical parameters of IVF-ET. Conclusions: No association was found between IVF-ET outcome and cervical infection, cytopathologic result, HPV detection, or result from the colposcopy or biopsy. Extensive testing and treatment for cervical infection do not appear necessary in IVF-ET candidates. © 2007 International Federation of Gynecology and Obstetrics. Published by Elsevier Ireland Ltd. All rights reserved.
1. Introduction Liquid-based cytologic screening, as with the thin-layer preparation cytologic test (TCT), offers advantages over the conventional cervical smear. It allows for a better microscopic visualization of exfoliated cells because there is less overlapping of epithelial cells and less obscuring from blood
⁎ Corresponding author. Reproductive Medical Center, Peking University Third Hospital, No. 49, North Gardern Road, Haidian District, Beijing, 100083, China. Tel.: +86 10 62017691 2056; fax: +86 10 62013283. E-mail address: [email protected] (Y. Wang).
or inflamed cells. Cells remaining in suspension after the screening test can be used for human papillomavirus (HPV) DNA testing, with the assurance that this (or other) ancillary study is being performed with an adequate specimen [1]. Screening with the TCT is done throughout China to prevent complications of infection. Abnormal cytologic results are followed by colposcopy and HPV testing, and the screening also detects the presence and severity of trichomonasis, moniliasis, bacterial vaginosis, and other infections. Most squamous cell carcinomas of the uterine cervix are related to HPV; and during the childbearing period untreated cervical intraepithelial neoplasia (CIN) may progress to invasive carcinoma, whose treatment in a pregnant woman would increase the risk of an
0020-7292/$ - see front matter © 2007 International Federation of Gynecology and Obstetrics. Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ijgo.2007.09.035
22 Table 1
Y. Wang et al. Table 3
General patient information
Characteristic
Pregnant group Nonpregnant group (n = 415) (n = 629)
Age, years Education Primary school Middle school High school College and higher Cause of infertilitya 1 2 3 4 5 Duration of infertility, years Type of infertility Primary Secondary
31.9 ± 3.6
32.6 ± 4.7
12 (2.9) 74 (17.8) 116 (28.0) 213 (51.3)
20 122 156 331
(3.2) (19.40) (24.80) (52.62)
226 (54.5) 133 (32.1) 15 (3.6) 20 (4.8) 21 (5.1) 4.7 ± 3.2
354 (56.28) 183 (29.09) 41 (6.52) 22 (3.50) 29 (4.61) 5.8 ± 4.4
204 (49.2) 211 (50.8)
368 (58.51) 261 (41.49)
Values are given as number (percentage) unless otherwise indicated. a 1 indicates a pathological tubal factor, 2 an abnormal spermiogram in the male partners, 3 an ovulation disorder, 4 endometriosis, 5 other factors.
undesired abortion [2]. On the other hand, there is a trend to overtreat these infections in China. Patients have sometimes been treated for several months at great cost, especially those undergoing in-vitro fertilization and embryo transfer (IVF-ET). The high prevalence of HPV infection and its association with tubal infertility and cervical cancer make this sexually transmitted disease of special interest. In a small percentage of infected women, an ascending infection will lead to pelvic inflammatory disease and ultimately to tubal infertility, which is the third most common cause of infertility in industrialized countries [3]. An increased rate of HPV infection is
Table 2
Patient distribution and IVF-ET outcome
Type of IVF and IVF-ET outcome
Pregnant group (n = 415)
Nonpregnant group (n = 629)
Total (n = 1044)
Regular IVF ICSI Sperm donation + IVF IVF-ETa 1 2 3 4
322 (77.6) 78 (18.8) 15 (3.6)
523 (83.1) 94 (14.9) 12 (1.9)
845 (80.9) 172 (16.4) 27 (2.6)
308 (74.2) 38 (9.2) 21 (5.1) 21 (2.0)
NA NA NA NA
308 (29.5) 38 (3.6) 21 (2.0) 48 (4.6)
ET embryo transfer, ICSI intracellular sperm injection, IVF invitro fertilization, NA not applicable. Values are given as number (percentage). a 1 indicates a singleton uterine pregnancy, 2 a spontaneous abortion, 3 an ectopic pregnancy, and 4 a twin or triplet pregnancy.
Condition detected
Influence of cervical lesions on IVF-ET outcome Pregnant group
Clinical sign of cervicitisa 1 43 (10.4) 2 215 (51.8) 3 100 (24.1) 4 57 (13.7) Infection on TCT None 317 (76.4) Mild 4 (0.9) Moderate 50 (12.1) Severe 44 (10.6)
Nonpregnant group
Total
53 368 135 73
(8.4) (58.5) (21.5) (11.6)
96 583 235 130
(9.2) (55.8) (22.5) (12.5)
435 7 108 79
(69.2) (1.1) (17.2 (12.6)
752 11 158 123
(72.0) (1.1) (15.1) (11.8)
TCT thin-layer preparation cytologic test. Values are given as number (percentage). a 1 indicates a smooth cervix, 2 a mild cervical erosion, 3 a moderate cervical erosion, and 4 a severe cervical erosion.
considered to reflect an increase in sexual activity in the general population [4]; and sexually active adolescents, who are at a high risk for STD, may be unaware that STD may cause infertility, and that treating infertility and severe cervical pathology could negatively affect family planning many years after the infections are cured. The infections detected via the TCT have not been investigated for their relationship to IVF-ET outcome. To do this we decided to determine what relationships, if any, existed in Chinese women between IVF-ET outcome and the cytologic abnormalities and various cervical infections revealed by the TCT.
2. Material and methods 2.1. Study participants This retrospective study was conducted with 1044 infertile Chinese women enrolled for IVF or intracellular sperm injection
Table 4 Detection of HPV and other infections on digital colposcopy and TCT in women undergoing IVF Method of evaluation Digital colposcopy Chronic cervicitis No iodine uptake Acetowhitening Abnormal angioarchitecture TCT ASCUS LSIL HSIL HPV testing
Pregnancy Nonpregnancy Total 3 3 4 4
(0.7) (0.7) (1.0) (1.0)
3 (0.5) 6 (1.0) 4 (0.6) 4 (0.6)
6 9 8 8
(0.6) (0.9) (0.8) (0.8)
11 8 2 4
(2.7) (1.9) (0.5) (1.0)
16 (2.5) 10 (1.6) 1 (0.2) 6 (1.0)
27 18 3 10
(2.6) (1.7) (0.3) (1.0)
ASCUS atypical squamous cells of undetermined significance, LSIL low-grade squamous intraepithelial lesion (including cervical intraepithelial neoplasia [CIN 1]), HSIL high-grade squamous intraepithelial lesion (including CIN 2 and CIN 3), TCT thin-layer preparation cytologic test. Values are given as number (percentage).
Relationship of cytopathology and cervical infection to in-vitro fertilization and embryo transfer
lesions (LSIL and HSIL) and those testing positive for HPV underwent a digital colposcopy and a cervical biopsy. Cells were layered on a glass slide and the brush was immersed in 5 mL of a phosphate-buffered saline solution. Cytologic results were classified using the 2001 Bethesda System (Terminology available at: http://bethesda2001.cancer.gov/terminology.html). This system classifies abnormal epithelial cells as atypical squamous cells of undetermined significance (ASCUS), LSIL (including CIN 1), HSIL (including CIN 2 and CIN 3), and squamous cell carcinoma.
Table 5 Cervical biopsy results in women undergoing invitro fertilization and embryo transfer Study group
Chronic Koilocytosis Glandular Condylomata cervicitis metaplasia acuminata
Pregnant 6 (1.5) Non 9 (1.4) pregnant Total 15 (1.4)
3 (0.7) 3 (0.5)
4 (1.0) 4 (0.6)
1 (0.2) 1 (0.2)
6 (0.6)
8 (0.8)
2 (0.2)
23
Values are given as number (percentage).
2.3. IVF-ET treatment at the Fertility Clinic of the Reproductive Medical Center of Peking University Third Hospital between January 1 and December 31, 2005. The main cause of sterility was tubal infertility or, in the male partner, abnormal semen. Study inclusion criteria were infertility for more than 1 year, age between 25 and 38 years, a morphologically normal uterus and cervix, good response to gonadotropins, and 2 or more goodquality embryos transferred (Table 1).
Cytologic screening was followed by a routine IVF-ET protocol. In IVF cycles, the long or short protocol was used for stimulation—i.e., gonadotropin-releasing hormone analogues (0.1 mg Diphereline; Ipsen, France) were administered for downregulation 5 to 7 days before the beginning of the cycle or on the second day of the cycle. Then all patients were treated with human menopausal gonadotropin (hMG; Livzon Pharmaceutical Group, Zhuhai, China) or recombinant follicle stimulating hormone (FSH; GONAL-f, Merck Serono, Switzerland) to stimulate ovulation on the third day of the cycle. Follicle diameters and serum concentrations of estradiol-17b (E2), progesterone (P) and luteinizing hormone (LH) were monitored starting Day 8 of the cycle. On the day when 3 follicles had a diameter greater than 17 mm, serum levels of E2, P, and LH were measured and 10,000 IU of human chorionic gonadotropin (hCG; Livzon Pharmaceutical
2.2. The TCT The ThinPrep Pap test (Cytyc, Marlborough, Massachusetts, USA) was used. Cervical scrapes were obtained with a cytobrush before ovarian stimulation with gonadotropins was started. Women with low- and high-grade squamous intraepithelial
Table 6
Relationship between TCT results and clinical parameters of IVF
Variable
Neoplasia subgroup
ASCUS (n = 27) IVF-ET outcome No. of follicles No. of oocytes No. of embryos transferred No. of goodquality embryos
Infection subgroups
LSIL (n = 18)
HSIL (n = 3)
HPV (n = 10)
Other than HPV Mild (n = 11)
Moderate (n = 158)
Severe (n = 123)
Normal (n = 694)
15.6 ± 6.2 13.5 ± 6.0 2.3 ± 0.5
15.4 ± 10.6 12.1 ± 6.4 2.4 ± 0.5
13.2 ± 8.8 11.5 ± 7.2 2.0 ± 0.8
11.6 ± 8.1 12.6 ± 6.8 1.9 ± 0.4
9.7 ± 8.0 13.7 ± 9.1 2.2 ± 0.4
10.9 ± 9.5 13.2 ± 7.2 2.0 ± 0.8
11.5 ± 8.7 13.5 ± 6.7 2.2 ± 0.7
15.5 ± 8.6 14.1 ± 8.0 2.3 ± 1.0
1.9 ± 1.0
2.3 ± 0.8
2.2 ± 0.5
1.9 ± 0.4
1.7 ± 1.0
1.9 ± 0.9
1.9 ± 0.8
1.9 ± 0.8
On the day of hCG administration Size of follicles, mm N 18 4.8 ± 2.0 4.0 ± 1.2 3.9 ± 1.4 5.0 ± 1.9 4.8 ± 1.7 4.8 ± 2.4 4.8 ± 2.1 4.8 ± 2.6 15–18 10.5 ± 2.9 5 ± 2.4 5.5 ± 3.8 4.1 ± 2.3 3.7 ± 2.8 4.9 ± 3.6 4.6 ± 2.3 4.7 ± 3.1 b 15 6.0 ± 2.1 8.6 ± 8.5 7.4 ± 6.5 5.4 ± 2.3 6.3 ± 4.9 5.9 ± 4.3 5.7 ± 3.3 6.1 ± 4.7 FSH, IU/L 16.9 ± 7.8 17.1 ± 6.8 18.1 ± 6.5 19.8 ± 4.8 20.2 ± 5.3 18.1 ± 6.7 18.6 ± 6.7 18.3 ± 7.3 hMG, 75 IU 19.5 ± 19.5 14.8 ± 5.0 14.8 ± 5.7 19.2 ± 16.4 25.0 ± 17.5 17.9 ± 14.3 20.1 ± 15.5 20.6 ± 19.3 7511.4 ± 6970 ± 7201.1 ± 6953.6 ± 8815.3 ± 8987.2 ± 9286.8 ± 8321.7 ± E2, pmol/L 4866.0 6027.1 3986.5 4245.4 6141.9 7657.6 7187.5 5484.6 P, nmol/L 2.3 ± 1.4 1.7 ± 1. 4 2.0 ± 1.6 2.4 ± 1.1 3.6 ± 1.9 2.6 ± 1.6 2.5 ± 1.7 2.7 ± 2.0 LH, IU/L 0.8 ± 0.6 1.7 ± 1.4 1.9 ± 1.5 0.69 ± 0.41 2.6 ± 2.2 1.2 ± 1.0 1.2 ± 1.0 1.7 ± 1.4 E2 estradiol, FSH follicle stimulating hormone (recombinant, administered), hCG human chorionic gonadotropin (administered), hMG human menopausal gonadotropin (administered), HPV human papillomavirus, IVF-ET in-vitro fertilization and embryo transfer, LH luteinizing hormone, P progesterone. Values are given as mean ± SD.
24 Group, Zhuhai, China) was injected at 8:00 PM. Thirty-six hours later, oocytes were harvested. If a positive result for HPV presence was obtained with the TCTand HPV testing, a digital colposcopic evaluation and a biopsy of the cervix were performed for further diagnosis. Colposcopic findings were graded as normal, abnormal, or cancerous. Colposcopic evidence of lesions included acetowhite areas, no or poor iodine uptake, abnormal angioarchitecture, punctation, mosaic pattern, open glands, and leukoplakia. Histologically, CIN 1 lesions were graded as LSIL and CIN 2 and CIN 3 lesions were both graded as HSIL.
2.4. Main outcome measures Pregnancy rate, numbers of follicles and retrieved oocytes, embryo quality and quantity, total amount of recombinant FSH (GONAL-f) and hMG administered, and serum levels of E2, P, and LH on the day of the hCG injection were compared among groups with different cervical lesions.
2.5. Statistical analyses Statistical analyses were performed using the SPSS software package, version 10.0 (SPSS, Chicago, Illinois, USA). The χ2 test was used to assess differences in categorical variables between the 2 groups. The Fischer exact test was used for 2 × 2 tables. P b 0.05 was considered statistically significant.
3. Results Following IVF-ET, the patients were assigned to the pregnant group (n = 415) or the nonpregnant group (n = 629) and matched by age; education; cause, duration, and type of infertility; and type of IVF (Tables 1 and 2). There were no statistically significant differences in pregnancy rates between women with disease and healthy women. No treatment for infection was administered in either group, and the groups did not significantly differ in clinical degree of cervicitis. Furthermore, as detected by the TCT, infection rates (aside from the rates of trichomonasis, moniliasis, and bacterial vaginosis) and cytopathologic severity were similar in the 2 groups. Likewise, neither HPV detection nor the results of the digital colposcopy and cervical biopsy were correlated with IVF-ET parameters (Tables 3, 4 and 5). The IVF-ET parameters (i.e., the numbers of follicles, retrieved oocytes, and transferable embryos; the quality of embryos; the total quantity of gonadotropins administered (GONAL-f and hMG); and the serum levels of E2, P, and LH on the day of the hCG injection were not found to be affected by the presence of ASCUS, LSIL, and/or HSIL, or by the presence of HPV or other infection, whether mild, moderate, or severe (Table 6).
4. Discussion The Bethesda System is the product of a 1988 National Cancer Institute workshop that enables laboratories to provide descriptive results reports which, in turn, effectively guide
Y. Wang et al. clinicians in their treatment decisions. Within 2 years, the system was adopted by most screening laboratories in the United States. The sensitivity of the Papanicolaou (Pap) test for identifying high-grade lesions, usually years before they become cancerous, is between 60% and 80% [5]. The Bethesda System is very useful to cytotechnologists who have to detect about a dozen abnormal cells among up to 100,000 cells on a slide. Today, along with the TCT, Pap tests are frequently combined in China, where more and more laboratories use the Bethesda System. In this study, the cytologic results were classified using the revised 2001 Bethesda System. Abnormal cytologic results are more frequently noted in women eligible for IVF treatment (6%) than in women from the general population (4%), and so is moderate or severe dyskaryosis [6]. In about 40% of women with abnormal Pap smears, infertility could still be explained by menstrual cycle problems, cervical factors, tubal and/or uterine factors, or endometriosis [6]. These results contrast with those of Lundqvist et al. [7], who reported abnormal cytologic results for 2% of women admitted for IVF and 4% of controls. All the women in the former group but only half of the controls were HPV positive in that study. The IVF treatment was successful in 33% of the patients with abnormal cervical cells and in 50% of those with HPV. The authors concluded that the prevalence of HPV among the infertile women in their series was a little lower than that which can be expected in a random sample of fertile women of similar age. Thus, it seems unlikely that HPV infection results in infertility. In that study, the pregnancy rate following IVF-ET was similar in women with and in those without HPV, and consequently HPV infection did not affect the outcome of the procedures [7]. The conclusion of our own study, in which the main causes of infertility were tubal factors or abnormal semen, is the same. Regarding the distribution of HPV infection in our study, there were no significant differences among patients with different causes of infertility and different levels of education. Although more women with tubal factor subfertility have been found to have mild or severe dyskaryosis or carcinomas in situ, the differences have not been found to be statistically significant [6]. In a matched series of Chinese women undergoing IVF-ET, we found that cervical infections (with trichomonas, moniliasis, and bacterial vaginosis left aside) and cervical cytopathology, all detected via the TCT, did not affect IVF-ET outcome even though these patients were not treated for their infections. Similarly, neither a diagnosis of HPV nor the results of the colposcopy and cervical biopsy were associated with IVF outcome. Genital HPV infection is sexually transmitted. In the present study the HPV tests were performed during an examination for infertility or in connection with oocyte retrieval. At that time the decision to try to fertilize the woman had already been made and the result of the HPV analysis did not interfere with the IVF procedure. There is no over-representation of HPV infection in infertile women. Nevertheless, it is quite clear that HPV screening should be included in the initial infertility work-up, so that abnormal findings could be treated before planning for IVF and pregnancy. A higher prevalence of severely abnormal cytologic findings has been noted among infertile women visiting outpatient clinics for IVF treatment than among the general female
Relationship of cytopathology and cervical infection to in-vitro fertilization and embryo transfer population in the same geographical area [6], and this observation may have led some authors to believe that STD is more frequent in infertile women. It has been suggested that an infection with Chlamydia trachomatis may increase the host's susceptibility to HPV infection or enhance the effects of HPV [8], and we could expect an association between C. trachomatis and cervical premalignancies, along with a higher frequency of abnormal cytologic results, in infertile women with tubal factors of infertility. However, we found the same distribution of abnormal cytologic results in women with other factors contributing to infertility, such as ovulation disorders, endometriosis, or a partner with abnormal semen; these results do not support the hypothesis of an association between C. trachomatis and cervical malignancies. Accumulated studies have shown that HPV is present in a substantial proportion of the sperm cells of male patients attending fertility clinics [9]. It should be emphasized that the partners of women with genital HPV infection are usually infected and it is important to evaluate the man, even if he is asymptomatic, not only as part of the infertility investigation but also to see if he is harboring HPV [10]. Further, HPV infection has been considered to be a cause of male infertility [11] and to affect sperm cell motility negatively; the incidence of asthenozoospermia appears associated with sperm harboring HPV [12]. In our study, neither infection with HPV, nor cytopathologic findings or cervical infection detected via the TCT was found to influence IVF-ET parameters. Steroid hormones have been identified as possible cofactors [13] for HPV gene expression. These hormones directly induce gene expression in HPV 16 and HPV 18 through hormone response elements in the regulatory region of the viral genome in human HeLa cells and cervical keratinocytes [14–16]. Moreover, HPV 16 transcription is enhanced by estrogen in vitro [17]. The longterm use of oral contraceptives increases the relative risk of developing condylomata acuminata [18] and oral contraceptives have been shown to interact with HPV to increase the risk of cervical cancer [19]. Infection with a high-risk HPV type was not associated with ovarian stimulation as measured by serial measurements of serum E2 levels, nor was it associated with an increased viral load or the increased detection of high-risk types [20]. The prevalence of high-risk types of HPV in the screening study was not significantly different from the prevalence in patients enrolled in the assisted reproductive technology program before stimulation. Furthermore, when women were divided into subgroups according to oral contraceptive use and reproductive technology used, no statistically significant differences were seen in those testing positive for high-risk HPV. Likewise, ovarian stimulation was not associated with increased detection of high-risk HPV types or increased viral load [20]. It has been shown in vitro that estrogen treatment of SiHa cells can induce viral replication [17], but viral load and the detection of high-risk HPV types were not influenced by stimulation with gonadotropins. A possible explanation is that the mean period of ovarian stimulation is short in women treated with assisted reproductive technology, and that the period of elevated E2 levels in these women may be too short to have a biological effect on HPV [20].
25
Cervical infections in IVF patients can be caused by the IVFET procedures themselves. Because these procedures involve needle puncture of the vagina and catheter placement of embryos through the cervix, contamination is possible from micro-organisms in the vagina and cervical canal. Infection of the oocyte or developing embryo could also impair embryo development and prevent the pregnancy from occurring [21]. In fact, the pregnancy rate is reduced by 50% when bacteria are isolated from the ETcatheter tip. Once in the endometrial cavity, virulent bacteria could stimulate macrophages and other immune cells and induce proinflammatory cytotoxic responses [21]. Such bacteria would attract neutrophils and injure the embryo or prevent implantation [21]. Despite satisfactory embryo morphology and adequate histologic and histochemical endometrial characteristics, overall pregnancy rates remain poor following IVF-ET. This indicates that additional factors might interfere with the normal embryo implantation process [22]. Evidence of inflammation following IVF-ET needs to be studied to further examine the hypothesis that inflammation is a mechanism by which bacteria may affect pregnancy outcome. Given the findings of the present study, we suggest to avoid overtreatment of cervical infections and cytopathologic abnormalities detected via the TCT in women undergoing IVF-ET, if these are the only adverse conditions identified.
References [1] Sherman ME. Chapter 11: future directions in cervical pathology. J Natl Cancer Inst Monogr 2003;31:72–9. [2] Nevin J, Soeters R, Dehaeck K, Bloch B, van-Wyk L. Cervical carcinoma associated with pregnancy. Obstet Gynecol Surv 1995;50: 228–39. [3] Hull MG, Glazener CM, Kelly NJ, Conway DI, Foster PA, Hinton RA, et al. Population study of causes, treatment, and outcome of infertility. Br Med J (Clin Res Ed) 1985;291:1693–7. [4] van Valkengoed I, Postma MJ, Morre SA, van den Brule AJ, Meijer CJ, Bouter LM, et al. Cost effectiveness analysis of a population based screening programme for asymptomatic Chlamydia trachomatis infections in women by means of home obtained urine specimens. Sex Transm Infect 2001;77: 276–82. [5] Twombly R. New Pap test terminology: management guidelines published. J Natl Cancer Inst 2002;94:878–80. [6] van Hamont D, Nissen LH, Siebers AG, Hendriks JC, Melchers WJ, Kremer JA, et al. Abnormal cervical cytology in women eligible for IVF. Hum Reprod 2006;21:2359–63. [7] Lundqvist M, Westin C, Lundkvist Ö, Simberg N, Strand A, Andersson S, et al. Cytologic screening and human papilloma virus test in women undergoing artificial fertilization. Acta Obstet Gynecol Scand 2002;81:949–53. [8] Castle PE, Hillier SL, Rabe LK, Hildesheim A, Herrero R, Bratti MC, et al. An association of cervical inflammation with high-grade cervical neoplasia in women infected with oncogenic human papillomavirus (HPV). Cancer Epidemiol Biomarkers Prev 2001;10: 1021–7. [9] Rintala MA, Pollanen PP, Nikkanen VP, Grenman SE, Syrjanen SM. Human papillomavirus DNA is found in the vas deferens. J Infect Dis 2002;185:1664–7. [10] Walboomers JM. Human papillomavirus detection and cervical cancer screening. Patologia 1999;32:270. [11] Rohde V, Erles K, Sattler HP, Derouet H, Wullich B, Schlehofer JR. Detection of adeno-associated virus in human semen: does viral
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[17] Mitrani-Rosenbaum S, Tsvieli R, Tur-Kaspa R. Oestrogen stimulates differential transcription of human papillomavirus type 16 in SiHa cervical carcinoma cells. J Gen Virol 1989;70: 2227–32. [18] Ross JD. Is oral contraceptive use associated with genital warts? Genitourin Med 1996;72:330–3. [19] Hildesheim A, Reeves WC, Brinton LA, Lavery C, Brenes M, De la Guardia ME. Association of oral contraceptive use and human papillomaviruses in invasive cervical cancers. Int J Cancer 1990;45: 860–4. [20] Strehler E, Sterzik K, Malthaner D, Hoyer H, Nindl I, Schneider A. Influence of ovarian stimulation on the detection of human papillomavirus DNA in cervical scrapes obtained from patients undergoing assisted reproductive techniques. Fertil Steril 1999;71: 815–20. [21] Moore DE, Soules MR, Klein NA, Fujimoto VY, Agnew KJ, Eschenbach DA. Bacteria in the transfer catheter tip influence the live-birth rate after in vitro fertilization. Fertil Steril 2000;74:1118–24. [22] Fanchin R, Harmas A, Benaoudia F, Lundkvist U, Olivennes F, Frydman R. Microbial flora of the cervix assessed at the time of embryo transfer adversely affects in vitro fertilization outcome. Fertil Steril 1998;70:866–70.
a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / i j g o
CLINICAL ARTICLE
Relationship of cytopathology and cervical infection to outcome of in-vitro fertilization and embryo transfer Ying Wang ⁎, Changjuan Wang, Jie Qiao, Lina Wang, Siqun Liang Reproductive Medical Center, Peking University Third Hospital, Beijing, China Received 31 July 2007; received in revised form 17 September 2007; accepted 25 September 2007
KEYWORDS Atypical squamous cells of undetermined significance; In-vitro fertilization and embryo transfer; Squamous intraepithelial lesion; Thin-layer preparation cytologic test
Abstract Objective: To determine whether a relationship exists between in-vitro fertilization and embryo transfer (IVF-ET) outcome and cervical infection or presence of human papillomavirus (HPV). Method: Cervical scrapes, digital colposcopies, and cervical biopsies were performed in 1044 Chinese women undergoing IVF for tubal infertility or, in their partners, abnormal semen. The pregnant (n = 415) and nonpregnant (n = 629) groups differed neither in clinical signs of cervical inflammation nor in rate of HPV detection. Results: There were no associations between IVF-ET outcome and infection rate, degree of cytopathologic abnormality, detection of HPV, or results of digital colposcopy and cervical biopsy. Cytologic results did not correlate with any of the clinical parameters of IVF-ET. Conclusions: No association was found between IVF-ET outcome and cervical infection, cytopathologic result, HPV detection, or result from the colposcopy or biopsy. Extensive testing and treatment for cervical infection do not appear necessary in IVF-ET candidates. © 2007 International Federation of Gynecology and Obstetrics. Published by Elsevier Ireland Ltd. All rights reserved.
1. Introduction Liquid-based cytologic screening, as with the thin-layer preparation cytologic test (TCT), offers advantages over the conventional cervical smear. It allows for a better microscopic visualization of exfoliated cells because there is less overlapping of epithelial cells and less obscuring from blood
⁎ Corresponding author. Reproductive Medical Center, Peking University Third Hospital, No. 49, North Gardern Road, Haidian District, Beijing, 100083, China. Tel.: +86 10 62017691 2056; fax: +86 10 62013283. E-mail address: [email protected] (Y. Wang).
or inflamed cells. Cells remaining in suspension after the screening test can be used for human papillomavirus (HPV) DNA testing, with the assurance that this (or other) ancillary study is being performed with an adequate specimen [1]. Screening with the TCT is done throughout China to prevent complications of infection. Abnormal cytologic results are followed by colposcopy and HPV testing, and the screening also detects the presence and severity of trichomonasis, moniliasis, bacterial vaginosis, and other infections. Most squamous cell carcinomas of the uterine cervix are related to HPV; and during the childbearing period untreated cervical intraepithelial neoplasia (CIN) may progress to invasive carcinoma, whose treatment in a pregnant woman would increase the risk of an
0020-7292/$ - see front matter © 2007 International Federation of Gynecology and Obstetrics. Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ijgo.2007.09.035
22 Table 1
Y. Wang et al. Table 3
General patient information
Characteristic
Pregnant group Nonpregnant group (n = 415) (n = 629)
Age, years Education Primary school Middle school High school College and higher Cause of infertilitya 1 2 3 4 5 Duration of infertility, years Type of infertility Primary Secondary
31.9 ± 3.6
32.6 ± 4.7
12 (2.9) 74 (17.8) 116 (28.0) 213 (51.3)
20 122 156 331
(3.2) (19.40) (24.80) (52.62)
226 (54.5) 133 (32.1) 15 (3.6) 20 (4.8) 21 (5.1) 4.7 ± 3.2
354 (56.28) 183 (29.09) 41 (6.52) 22 (3.50) 29 (4.61) 5.8 ± 4.4
204 (49.2) 211 (50.8)
368 (58.51) 261 (41.49)
Values are given as number (percentage) unless otherwise indicated. a 1 indicates a pathological tubal factor, 2 an abnormal spermiogram in the male partners, 3 an ovulation disorder, 4 endometriosis, 5 other factors.
undesired abortion [2]. On the other hand, there is a trend to overtreat these infections in China. Patients have sometimes been treated for several months at great cost, especially those undergoing in-vitro fertilization and embryo transfer (IVF-ET). The high prevalence of HPV infection and its association with tubal infertility and cervical cancer make this sexually transmitted disease of special interest. In a small percentage of infected women, an ascending infection will lead to pelvic inflammatory disease and ultimately to tubal infertility, which is the third most common cause of infertility in industrialized countries [3]. An increased rate of HPV infection is
Table 2
Patient distribution and IVF-ET outcome
Type of IVF and IVF-ET outcome
Pregnant group (n = 415)
Nonpregnant group (n = 629)
Total (n = 1044)
Regular IVF ICSI Sperm donation + IVF IVF-ETa 1 2 3 4
322 (77.6) 78 (18.8) 15 (3.6)
523 (83.1) 94 (14.9) 12 (1.9)
845 (80.9) 172 (16.4) 27 (2.6)
308 (74.2) 38 (9.2) 21 (5.1) 21 (2.0)
NA NA NA NA
308 (29.5) 38 (3.6) 21 (2.0) 48 (4.6)
ET embryo transfer, ICSI intracellular sperm injection, IVF invitro fertilization, NA not applicable. Values are given as number (percentage). a 1 indicates a singleton uterine pregnancy, 2 a spontaneous abortion, 3 an ectopic pregnancy, and 4 a twin or triplet pregnancy.
Condition detected
Influence of cervical lesions on IVF-ET outcome Pregnant group
Clinical sign of cervicitisa 1 43 (10.4) 2 215 (51.8) 3 100 (24.1) 4 57 (13.7) Infection on TCT None 317 (76.4) Mild 4 (0.9) Moderate 50 (12.1) Severe 44 (10.6)
Nonpregnant group
Total
53 368 135 73
(8.4) (58.5) (21.5) (11.6)
96 583 235 130
(9.2) (55.8) (22.5) (12.5)
435 7 108 79
(69.2) (1.1) (17.2 (12.6)
752 11 158 123
(72.0) (1.1) (15.1) (11.8)
TCT thin-layer preparation cytologic test. Values are given as number (percentage). a 1 indicates a smooth cervix, 2 a mild cervical erosion, 3 a moderate cervical erosion, and 4 a severe cervical erosion.
considered to reflect an increase in sexual activity in the general population [4]; and sexually active adolescents, who are at a high risk for STD, may be unaware that STD may cause infertility, and that treating infertility and severe cervical pathology could negatively affect family planning many years after the infections are cured. The infections detected via the TCT have not been investigated for their relationship to IVF-ET outcome. To do this we decided to determine what relationships, if any, existed in Chinese women between IVF-ET outcome and the cytologic abnormalities and various cervical infections revealed by the TCT.
2. Material and methods 2.1. Study participants This retrospective study was conducted with 1044 infertile Chinese women enrolled for IVF or intracellular sperm injection
Table 4 Detection of HPV and other infections on digital colposcopy and TCT in women undergoing IVF Method of evaluation Digital colposcopy Chronic cervicitis No iodine uptake Acetowhitening Abnormal angioarchitecture TCT ASCUS LSIL HSIL HPV testing
Pregnancy Nonpregnancy Total 3 3 4 4
(0.7) (0.7) (1.0) (1.0)
3 (0.5) 6 (1.0) 4 (0.6) 4 (0.6)
6 9 8 8
(0.6) (0.9) (0.8) (0.8)
11 8 2 4
(2.7) (1.9) (0.5) (1.0)
16 (2.5) 10 (1.6) 1 (0.2) 6 (1.0)
27 18 3 10
(2.6) (1.7) (0.3) (1.0)
ASCUS atypical squamous cells of undetermined significance, LSIL low-grade squamous intraepithelial lesion (including cervical intraepithelial neoplasia [CIN 1]), HSIL high-grade squamous intraepithelial lesion (including CIN 2 and CIN 3), TCT thin-layer preparation cytologic test. Values are given as number (percentage).
Relationship of cytopathology and cervical infection to in-vitro fertilization and embryo transfer
lesions (LSIL and HSIL) and those testing positive for HPV underwent a digital colposcopy and a cervical biopsy. Cells were layered on a glass slide and the brush was immersed in 5 mL of a phosphate-buffered saline solution. Cytologic results were classified using the 2001 Bethesda System (Terminology available at: http://bethesda2001.cancer.gov/terminology.html). This system classifies abnormal epithelial cells as atypical squamous cells of undetermined significance (ASCUS), LSIL (including CIN 1), HSIL (including CIN 2 and CIN 3), and squamous cell carcinoma.
Table 5 Cervical biopsy results in women undergoing invitro fertilization and embryo transfer Study group
Chronic Koilocytosis Glandular Condylomata cervicitis metaplasia acuminata
Pregnant 6 (1.5) Non 9 (1.4) pregnant Total 15 (1.4)
3 (0.7) 3 (0.5)
4 (1.0) 4 (0.6)
1 (0.2) 1 (0.2)
6 (0.6)
8 (0.8)
2 (0.2)
23
Values are given as number (percentage).
2.3. IVF-ET treatment at the Fertility Clinic of the Reproductive Medical Center of Peking University Third Hospital between January 1 and December 31, 2005. The main cause of sterility was tubal infertility or, in the male partner, abnormal semen. Study inclusion criteria were infertility for more than 1 year, age between 25 and 38 years, a morphologically normal uterus and cervix, good response to gonadotropins, and 2 or more goodquality embryos transferred (Table 1).
Cytologic screening was followed by a routine IVF-ET protocol. In IVF cycles, the long or short protocol was used for stimulation—i.e., gonadotropin-releasing hormone analogues (0.1 mg Diphereline; Ipsen, France) were administered for downregulation 5 to 7 days before the beginning of the cycle or on the second day of the cycle. Then all patients were treated with human menopausal gonadotropin (hMG; Livzon Pharmaceutical Group, Zhuhai, China) or recombinant follicle stimulating hormone (FSH; GONAL-f, Merck Serono, Switzerland) to stimulate ovulation on the third day of the cycle. Follicle diameters and serum concentrations of estradiol-17b (E2), progesterone (P) and luteinizing hormone (LH) were monitored starting Day 8 of the cycle. On the day when 3 follicles had a diameter greater than 17 mm, serum levels of E2, P, and LH were measured and 10,000 IU of human chorionic gonadotropin (hCG; Livzon Pharmaceutical
2.2. The TCT The ThinPrep Pap test (Cytyc, Marlborough, Massachusetts, USA) was used. Cervical scrapes were obtained with a cytobrush before ovarian stimulation with gonadotropins was started. Women with low- and high-grade squamous intraepithelial
Table 6
Relationship between TCT results and clinical parameters of IVF
Variable
Neoplasia subgroup
ASCUS (n = 27) IVF-ET outcome No. of follicles No. of oocytes No. of embryos transferred No. of goodquality embryos
Infection subgroups
LSIL (n = 18)
HSIL (n = 3)
HPV (n = 10)
Other than HPV Mild (n = 11)
Moderate (n = 158)
Severe (n = 123)
Normal (n = 694)
15.6 ± 6.2 13.5 ± 6.0 2.3 ± 0.5
15.4 ± 10.6 12.1 ± 6.4 2.4 ± 0.5
13.2 ± 8.8 11.5 ± 7.2 2.0 ± 0.8
11.6 ± 8.1 12.6 ± 6.8 1.9 ± 0.4
9.7 ± 8.0 13.7 ± 9.1 2.2 ± 0.4
10.9 ± 9.5 13.2 ± 7.2 2.0 ± 0.8
11.5 ± 8.7 13.5 ± 6.7 2.2 ± 0.7
15.5 ± 8.6 14.1 ± 8.0 2.3 ± 1.0
1.9 ± 1.0
2.3 ± 0.8
2.2 ± 0.5
1.9 ± 0.4
1.7 ± 1.0
1.9 ± 0.9
1.9 ± 0.8
1.9 ± 0.8
On the day of hCG administration Size of follicles, mm N 18 4.8 ± 2.0 4.0 ± 1.2 3.9 ± 1.4 5.0 ± 1.9 4.8 ± 1.7 4.8 ± 2.4 4.8 ± 2.1 4.8 ± 2.6 15–18 10.5 ± 2.9 5 ± 2.4 5.5 ± 3.8 4.1 ± 2.3 3.7 ± 2.8 4.9 ± 3.6 4.6 ± 2.3 4.7 ± 3.1 b 15 6.0 ± 2.1 8.6 ± 8.5 7.4 ± 6.5 5.4 ± 2.3 6.3 ± 4.9 5.9 ± 4.3 5.7 ± 3.3 6.1 ± 4.7 FSH, IU/L 16.9 ± 7.8 17.1 ± 6.8 18.1 ± 6.5 19.8 ± 4.8 20.2 ± 5.3 18.1 ± 6.7 18.6 ± 6.7 18.3 ± 7.3 hMG, 75 IU 19.5 ± 19.5 14.8 ± 5.0 14.8 ± 5.7 19.2 ± 16.4 25.0 ± 17.5 17.9 ± 14.3 20.1 ± 15.5 20.6 ± 19.3 7511.4 ± 6970 ± 7201.1 ± 6953.6 ± 8815.3 ± 8987.2 ± 9286.8 ± 8321.7 ± E2, pmol/L 4866.0 6027.1 3986.5 4245.4 6141.9 7657.6 7187.5 5484.6 P, nmol/L 2.3 ± 1.4 1.7 ± 1. 4 2.0 ± 1.6 2.4 ± 1.1 3.6 ± 1.9 2.6 ± 1.6 2.5 ± 1.7 2.7 ± 2.0 LH, IU/L 0.8 ± 0.6 1.7 ± 1.4 1.9 ± 1.5 0.69 ± 0.41 2.6 ± 2.2 1.2 ± 1.0 1.2 ± 1.0 1.7 ± 1.4 E2 estradiol, FSH follicle stimulating hormone (recombinant, administered), hCG human chorionic gonadotropin (administered), hMG human menopausal gonadotropin (administered), HPV human papillomavirus, IVF-ET in-vitro fertilization and embryo transfer, LH luteinizing hormone, P progesterone. Values are given as mean ± SD.
24 Group, Zhuhai, China) was injected at 8:00 PM. Thirty-six hours later, oocytes were harvested. If a positive result for HPV presence was obtained with the TCTand HPV testing, a digital colposcopic evaluation and a biopsy of the cervix were performed for further diagnosis. Colposcopic findings were graded as normal, abnormal, or cancerous. Colposcopic evidence of lesions included acetowhite areas, no or poor iodine uptake, abnormal angioarchitecture, punctation, mosaic pattern, open glands, and leukoplakia. Histologically, CIN 1 lesions were graded as LSIL and CIN 2 and CIN 3 lesions were both graded as HSIL.
2.4. Main outcome measures Pregnancy rate, numbers of follicles and retrieved oocytes, embryo quality and quantity, total amount of recombinant FSH (GONAL-f) and hMG administered, and serum levels of E2, P, and LH on the day of the hCG injection were compared among groups with different cervical lesions.
2.5. Statistical analyses Statistical analyses were performed using the SPSS software package, version 10.0 (SPSS, Chicago, Illinois, USA). The χ2 test was used to assess differences in categorical variables between the 2 groups. The Fischer exact test was used for 2 × 2 tables. P b 0.05 was considered statistically significant.
3. Results Following IVF-ET, the patients were assigned to the pregnant group (n = 415) or the nonpregnant group (n = 629) and matched by age; education; cause, duration, and type of infertility; and type of IVF (Tables 1 and 2). There were no statistically significant differences in pregnancy rates between women with disease and healthy women. No treatment for infection was administered in either group, and the groups did not significantly differ in clinical degree of cervicitis. Furthermore, as detected by the TCT, infection rates (aside from the rates of trichomonasis, moniliasis, and bacterial vaginosis) and cytopathologic severity were similar in the 2 groups. Likewise, neither HPV detection nor the results of the digital colposcopy and cervical biopsy were correlated with IVF-ET parameters (Tables 3, 4 and 5). The IVF-ET parameters (i.e., the numbers of follicles, retrieved oocytes, and transferable embryos; the quality of embryos; the total quantity of gonadotropins administered (GONAL-f and hMG); and the serum levels of E2, P, and LH on the day of the hCG injection were not found to be affected by the presence of ASCUS, LSIL, and/or HSIL, or by the presence of HPV or other infection, whether mild, moderate, or severe (Table 6).
4. Discussion The Bethesda System is the product of a 1988 National Cancer Institute workshop that enables laboratories to provide descriptive results reports which, in turn, effectively guide
Y. Wang et al. clinicians in their treatment decisions. Within 2 years, the system was adopted by most screening laboratories in the United States. The sensitivity of the Papanicolaou (Pap) test for identifying high-grade lesions, usually years before they become cancerous, is between 60% and 80% [5]. The Bethesda System is very useful to cytotechnologists who have to detect about a dozen abnormal cells among up to 100,000 cells on a slide. Today, along with the TCT, Pap tests are frequently combined in China, where more and more laboratories use the Bethesda System. In this study, the cytologic results were classified using the revised 2001 Bethesda System. Abnormal cytologic results are more frequently noted in women eligible for IVF treatment (6%) than in women from the general population (4%), and so is moderate or severe dyskaryosis [6]. In about 40% of women with abnormal Pap smears, infertility could still be explained by menstrual cycle problems, cervical factors, tubal and/or uterine factors, or endometriosis [6]. These results contrast with those of Lundqvist et al. [7], who reported abnormal cytologic results for 2% of women admitted for IVF and 4% of controls. All the women in the former group but only half of the controls were HPV positive in that study. The IVF treatment was successful in 33% of the patients with abnormal cervical cells and in 50% of those with HPV. The authors concluded that the prevalence of HPV among the infertile women in their series was a little lower than that which can be expected in a random sample of fertile women of similar age. Thus, it seems unlikely that HPV infection results in infertility. In that study, the pregnancy rate following IVF-ET was similar in women with and in those without HPV, and consequently HPV infection did not affect the outcome of the procedures [7]. The conclusion of our own study, in which the main causes of infertility were tubal factors or abnormal semen, is the same. Regarding the distribution of HPV infection in our study, there were no significant differences among patients with different causes of infertility and different levels of education. Although more women with tubal factor subfertility have been found to have mild or severe dyskaryosis or carcinomas in situ, the differences have not been found to be statistically significant [6]. In a matched series of Chinese women undergoing IVF-ET, we found that cervical infections (with trichomonas, moniliasis, and bacterial vaginosis left aside) and cervical cytopathology, all detected via the TCT, did not affect IVF-ET outcome even though these patients were not treated for their infections. Similarly, neither a diagnosis of HPV nor the results of the colposcopy and cervical biopsy were associated with IVF outcome. Genital HPV infection is sexually transmitted. In the present study the HPV tests were performed during an examination for infertility or in connection with oocyte retrieval. At that time the decision to try to fertilize the woman had already been made and the result of the HPV analysis did not interfere with the IVF procedure. There is no over-representation of HPV infection in infertile women. Nevertheless, it is quite clear that HPV screening should be included in the initial infertility work-up, so that abnormal findings could be treated before planning for IVF and pregnancy. A higher prevalence of severely abnormal cytologic findings has been noted among infertile women visiting outpatient clinics for IVF treatment than among the general female
Relationship of cytopathology and cervical infection to in-vitro fertilization and embryo transfer population in the same geographical area [6], and this observation may have led some authors to believe that STD is more frequent in infertile women. It has been suggested that an infection with Chlamydia trachomatis may increase the host's susceptibility to HPV infection or enhance the effects of HPV [8], and we could expect an association between C. trachomatis and cervical premalignancies, along with a higher frequency of abnormal cytologic results, in infertile women with tubal factors of infertility. However, we found the same distribution of abnormal cytologic results in women with other factors contributing to infertility, such as ovulation disorders, endometriosis, or a partner with abnormal semen; these results do not support the hypothesis of an association between C. trachomatis and cervical malignancies. Accumulated studies have shown that HPV is present in a substantial proportion of the sperm cells of male patients attending fertility clinics [9]. It should be emphasized that the partners of women with genital HPV infection are usually infected and it is important to evaluate the man, even if he is asymptomatic, not only as part of the infertility investigation but also to see if he is harboring HPV [10]. Further, HPV infection has been considered to be a cause of male infertility [11] and to affect sperm cell motility negatively; the incidence of asthenozoospermia appears associated with sperm harboring HPV [12]. In our study, neither infection with HPV, nor cytopathologic findings or cervical infection detected via the TCT was found to influence IVF-ET parameters. Steroid hormones have been identified as possible cofactors [13] for HPV gene expression. These hormones directly induce gene expression in HPV 16 and HPV 18 through hormone response elements in the regulatory region of the viral genome in human HeLa cells and cervical keratinocytes [14–16]. Moreover, HPV 16 transcription is enhanced by estrogen in vitro [17]. The longterm use of oral contraceptives increases the relative risk of developing condylomata acuminata [18] and oral contraceptives have been shown to interact with HPV to increase the risk of cervical cancer [19]. Infection with a high-risk HPV type was not associated with ovarian stimulation as measured by serial measurements of serum E2 levels, nor was it associated with an increased viral load or the increased detection of high-risk types [20]. The prevalence of high-risk types of HPV in the screening study was not significantly different from the prevalence in patients enrolled in the assisted reproductive technology program before stimulation. Furthermore, when women were divided into subgroups according to oral contraceptive use and reproductive technology used, no statistically significant differences were seen in those testing positive for high-risk HPV. Likewise, ovarian stimulation was not associated with increased detection of high-risk HPV types or increased viral load [20]. It has been shown in vitro that estrogen treatment of SiHa cells can induce viral replication [17], but viral load and the detection of high-risk HPV types were not influenced by stimulation with gonadotropins. A possible explanation is that the mean period of ovarian stimulation is short in women treated with assisted reproductive technology, and that the period of elevated E2 levels in these women may be too short to have a biological effect on HPV [20].
25
Cervical infections in IVF patients can be caused by the IVFET procedures themselves. Because these procedures involve needle puncture of the vagina and catheter placement of embryos through the cervix, contamination is possible from micro-organisms in the vagina and cervical canal. Infection of the oocyte or developing embryo could also impair embryo development and prevent the pregnancy from occurring [21]. In fact, the pregnancy rate is reduced by 50% when bacteria are isolated from the ETcatheter tip. Once in the endometrial cavity, virulent bacteria could stimulate macrophages and other immune cells and induce proinflammatory cytotoxic responses [21]. Such bacteria would attract neutrophils and injure the embryo or prevent implantation [21]. Despite satisfactory embryo morphology and adequate histologic and histochemical endometrial characteristics, overall pregnancy rates remain poor following IVF-ET. This indicates that additional factors might interfere with the normal embryo implantation process [22]. Evidence of inflammation following IVF-ET needs to be studied to further examine the hypothesis that inflammation is a mechanism by which bacteria may affect pregnancy outcome. Given the findings of the present study, we suggest to avoid overtreatment of cervical infections and cytopathologic abnormalities detected via the TCT in women undergoing IVF-ET, if these are the only adverse conditions identified.
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