Association Between Leptotrichia Amnionii and Atopobium Vaginae as A Risk Factor For Miscarriage States in A Specimens of Iraqi Women

Miscarriage is one of the fundamental complications occurring in pregnant women. Many fastidious and uncultivated bacterial species are related to miscarriage and have a significant role in the infection. The association between inverse pregnancy outcomes and infections of abnormal bacteria has been rarely investigated. Therefore, this study aimed to determine Leptotrichia amnionii and Atopobium vaginae as a risk factor for miscarriage states. A total of 80 cervical swabs and blood samples were obtained from females (34 non-pregnant with recurrent spontaneous miscarriage, 11 pregnant who had previous miscarriage, and 35 without miscarriage as control) who were referred to a hospital in Baghdad city. The relationship between demographic characteristics and miscarriage groups indicates a positive association between the age of 30 years and >45 years and miscarriage states, while no significant association (p>0.05) was found for the other parameters compared with the control. Also, no association was observed between the levels of TORCH IgM , antiphospholipid (APL) IgM, and anticardiolipidin (ACL) IgM antibodies and spontaneous miscarriage. The findings of RT-PCR detection revealed that, out of 45 samples of recurrent spontaneous miscarriage, 17 were detected with high-risk of L. aminonia compared with two samples from the control group. Also, 7 samples of recurrent spontaneous miscarriage were shown to be infected with A. vaginae compared with five samples from the control group. The adjusted odd ratios (ORs) of L. anmionii for spontaneous miscarriage and pregnancy states were 7.89 (CI 95%, 1.60 39.00) and 9.43 (CI 95%, 1.43 61.9), respectively, with highly significant difference between the study groups compared with the control. While no significant result was recorded between the adjusted ORs of A. vaginae for spontaneous miscarriage and pregnancy states, which were 1.23 (CI 95%, 0.416 3.67) and 0.636 (CI 95%, 0.083 4.88), respectively, compared with the control. Keywords; spontaneous miscarriage, Leptotrichia amnionii, Atopobium vaginae immunological factors نيب ةقلاعلا ايرتكب leptotrichia amnionii و Atopobium vaginae يف ةروطخ لماعك ءاسنلا نم ةنيع يف ضاهجلاا تلااح رعلا ا ايق ت نا رفغ رئاث ركا م , لادنق فيزوج ىهن ةيئايحلاا تايشقتلا مدق ، مهمعلا ةيمك ، دادغب ةعماج ، قا رعلا, دادغب ISSN: 0067-2904 Akram and Kandala Iraqi Journal of Science, 2021, Vol. 62, No. 1, pp: 83-95 84 ةصلاخلا .لماهحلا ءادشلا دشع ثدحت يتلا ةيساسلأا تافعازسلا دحأ هى ضايجلإا ايرتكبلا عاهنأ نم ديدعلا طبترت ةيوذغت تابمطتم ىلا جاتحت يتلا ةدقعم و ةبهعصو لاا يف رزتس ا ضايجلإاب ع نهكي يتلاو يف ميم رود ايل ىودعلا و ةردن ةجيتن يدكعلا لسحلا جئاتن نيب ةقلاعلا ةسا رد يف قمعتت يتلا ثاحبلاا ريغ ةيريتكبلا تابايتللااو ةيعيبطلا ىلإ ةسا ردلا هذى تفدى اذل . ديدحت ايرتكب ـل ـ leptotrichia amnionii و Atopobium vaginae طخ لماعك يف ةره حدم عسج مت .ضايجلإا تلااح تا نم محرلا قشع مد تاشيعو نم 08 نا ىث ( 43 ةشيع تلااح نم رركتسلا يئاقمتلا ضايجلإا يف و ، لماهحلا ريغ ءادشلا 11 تلااح مييدل لماهحلا ءادشلا نم ةقباس ضايجا و 43 ضايجإ نود ةأ رما ) )لماهح ريغ ةعهسجسك ةشيدم يف ىفذتدم نم ايعسج مت )ةرطيس راسعلأا نيب ةيباجيإ ةقلاع دهجو ىلإ ضايجلإا تاعهسجمو ةيفا رغهسيدلا صئارخلا نيب ةقلاعلا ريذت .دادغب يتلا نيب تحوا رت 48 ربكأ وأ امًاع و لا اسشيب ، ضايجلإا تلااح ت دجه ةيهشعم ةقلاع نيب ريياعسلا ىرخلأا ةسا ردلا ايتشسزت يتلا اقم اب ةنر ةرطيدل . تا رابتخلا ةبلاس ةجيتن تريظا كلذكو ةدازسلا مادجلأل ا ـل TORCH IgM ةدازسلا مادجلأاو ، IgM ديبيلهفسهفمل (APL) ةدازسلا مادجلأاو IgM نيدبيلهيدراكمل (ACL) مدعو ، طبا رتلا عم جم عيما .يئاقمتلا ضايجلإا و تشيب فذكلا جئاتن لاسعتساب RT-PCR نم ونأ نيب 08 ، ةشيع تريظا 11 ةشيع دجاهت L. aminonia لماعك نم نيتشيع عم ةنراقم ةيلاع ةرهطخ ةعهسجم ةرطيدلا , نيح يف تريظ A. vaginae يف 1 نم تاشيع عم ةنراقم رركتسلا يئاقمتلا ضايجلإا تاعهسجم ةعهسجم نم تاشيع سسخ ةرطيدلا . تريظا اسشيب L. anmionii ذا ا ريبك ايهشعم اطبا رت ب ميق تغم ORS (CI 95% 61.9, 1.43) 9.43 (CI 95%,-61.91.43)7.89 ضايجلإل ةنراقم لسحلاو يئاقمتلا ةعهسجم عم ، ةرطيدلا نيح يف هكي مل ن ـلا جئاتن يف يهشعم طبا رت كاشى A. vagina ميق تغمب امدشع ORS (CI 95%,-3.670.416)1.23 و (CI 95% 4.88, -0.083) 0.636 يئاقمتلا ضايجلإل .لسحلاو Introduction Miscarriage refers to pregnancy loss at less than 20 weeks of gestation; it is the most common complication of pregnancy. Several etiologies factors such as anatomic, genetic, endocrine, hematological and immunological factors have been reported to associate with this destructive complication of pregnancy [1, 2]. Since the word “abortion” is associated with elective termination, the term “miscarriage” is synonymous and often used with patients [3]. First-trimester miscarriage refers to the early stage in the course of a miscarriage when intact gestational sacs are still present within the uterine cavity, blighted ovum; missed, delayed, silent miscarriage, or early fetal demise. The diagnosis of early fetal demise is based either on the absence of cardiac activity in a visible embryo or on the absence of an embryo within a gestational sac [4]. Second-trimester pregnancy loss is defined as pregnancy loss after the 14th week and before the 24th week of gestation. However, it has been associated with infection, cervical insufficiency, uterine malformations, gene polymorphisms, fetal and placental anomalies, and genetic and acquired thrombophilia [5]. Recurrent spontaneous miscarriage (RSM) is described as the occurrence of 3 or more consecutive spontaneous miscarriages, in a series of conditions that lead to or could terminate in reproductive failure [6]. Recurrent spontaneous miscarriage is multifactorial, so that approximately half of all cases will remain unexplained. At present, there exist a small number of accepted etiologies for RSM. These include uncontrolled diabetes mellitus, untreated hypothyroidism, parental chromosomal abnormalities, anti-phospholipid antibody syndrome (APS), and certain uterine anatomic abnormalities. Other probable or possible etiologies include heritable and/or acquired thrombophilia, immunologic abnormalities, additional endocrine disorders, environmental factors, and infection [7, 8]. The correlation between the adverse outcomes of pregnancy and host infections was poorly examined, whereas intrauterine infections that occur in early and late pregnancy leading to a spontaneous miscarriage and spontaneous preterm birth were recorded [9]. Since the main route of placenta and fetus infection is from the vagina and cervix, most studies have attempted to find a link between miscarriage and abnormal bacterial flora in the lower genital tract [10]. However, bacterial vaginosis was reported to be causative for second-trimester miscarriage or premature delivery in the Akram and Kandala Iraqi Journal of Science, 2021, Vol. 62, No. 1, pp: 83-95 85 first trimester of pregnancy [11], although bacterial vaginosis-related bacteria and their toxins can cross the placenta and cause fetal complications [12]. Atopobium vaginaean bacteria are anaerobic, Gram-positive, non-mobile, sporogenous and noncapsulated, measuring approximately 0.6-0.9 μm and forming small clear colonies similar to small pinheads in Columbia blood agar cultures at 37°C [13]. In addition to the highlighted position in BV, A. vaginae infection and a higher risk of premature birth was associated [14]. Leptotrichia species are fastidious gram-negative, non-motile, long, fusiform rods, non-spore forming, anaerobic bacteria that exist as part of microorganisms of the human oral cavity and female genitalia [15]. A Leptotrichia sp. was first reported in 2002, when Shukla et al. [16] described L. amnionii as a novel causative species of infection in the female urogenital tract. Also, L. amnionii was isolated from the cervix of a patient with a premature rupture of the fetus membranes [17]. PCR analysis has been useful in detecting infections in patients with premature labour, intact membranes, and intra-amniotic infections compared to normal microbial cultures [18]. However, epidemiological data on the potential association between cervical microbiota and spontaneous miscarriage has been rarely reported. The objective of this study was to determine the cervical bacteria in different miscarriage samples using qPCR technique and investigate the correlation between spontaneous miscarriage and cervical bacteria in a sample of Iraqi women. Materials and Methods The Subjects All samples required in this study were collected from women (age ranged 18-51 years) attended as outpatients of gynecology department of the Medical City Teaching Hospital. Patient’s samples were selected according to questionnaire information, history of spontaneous abortion, and number of abortions (and other categories). The study included 80 females, 45 suffering a recurrent spontaneous miscarriage and 35 normal). This study was approved by the Ethics and Research Committee of the hospital under the supervision of the consultant, while approval for the sampling was obtained from patients and controls. Specimen collection Two types of samples were collected from women; first, 80 cervical swabs from the endocervical region after inserting a sterile speculum into the vagina. Second, blood samples (5 of venous blood) which were then transferred into polyethylene tubes, allowed to clot, and centrifuged at 3000 rpm for 10 minutes. The obtained clear serum was frozen at 20 oC until used for further immunological assays. Immunochromatography detection of antibodies In order to detect some microorganisms that maybe related to miscarriage in women, TORCH is a rapid test kit used in this study for the qualitative detection of TOX-IgM, RU-IgM, CMV-IgM, HSV2-IgM, and HSV-1-IgM antibodies in human serum. The procedure followed the manufacturer’s instructions (TORCH rapid test kit, Weifang Kanghua Biotech\China) [19] Determination of Anticardiolipin IgM and Antiphospholipid IgM antibodies The detection of antiphospholipid (APL) IgM and anticardiolipidin(ACL) IgM antibodies in the serum by was conducted using ELISA Kit, following the manufacturer’s instructions (Antiphospholipid and Anticardiolipin antibodies ELISA Kit, Cortez \ USA) [20, 21]. Extraction of DNA Total DNA (genomic and bacterial DNA) was isolated from cervical swab samples for molecular studies following the standard protocol for extraction. Genomic DNA isolation was achieved by using a DNA-sorb-A nucleic acid extraction kit. Nanodrop instrument (Nas-99/China) was used to determine DNA concentration and purity. The integrity of DNA was estimated by agarose gel electrophoresis. Real -Time Polymerase Chain Reaction For the qualitative detection of Leptotrichia amnionii and Atopobium vaginae Real Timefor L. amnionii and A. Vaginae were was conducted by using 16S rRNA primers (Table-1). The reaction mixture consisted of 5μl DNA template, 0.8μl of each primer, 1.25μl CXR dye, 12.5μl of Bright Green Express 1X qPCR Master mix, and 4.65μl Nuclease-free water, in a total volume of 25μl. The template DNA was amplified for 40 cycles of denaturation for 1 minute at 95°C, annealing of primers at 63°C for A. vaginae and 64°C for L. amnionii for 1 minute, and extension at 72°C for 1 minute. Fluorescent data were acquired during each extension phase [22]. Akram and Kandala Iraqi Journal of Science, 2021, Vol. 62, No. 1, pp: 83-95 86 Table 1The Oligonucleotide Sequences of Primers for RT-qPCR Detection of L. amnionii and A. vaginae The name of gene Primer sequence 5’ → 3’ size product 16S rRNA/ A.vaginae F-GCAGGGACGAGGCCGCAA R-GTGTTTCCACTGCTTCACCTAA 558 16S rRNA for L.amnionii F-CAATTCTGTGTGTGTGAAGAAG R-ACAGTTTTGTAGGCAAGCCTAT 229 Statistical Analysis The SPSS software (V 20.0) was utilized to estimate the effects of different factors on the study parameters. The correlation amongst the parameters was estimated using Pearson chi-squared (R) to test for significant differences at p< 0.05. Chi-squared and ANOVA tests were used to assess the differences in the categorical and continuous variables, respectively, among miscarriage patients. Results and Discussion A total of 80 specimens of endocervical cytobrush and blood were collected from both patients and control groups by a specialist physician at the gynecology department of the Medical City Teaching Hospital. These samples were divided into two groups. First, the patients group consisting of 45 women suffering a spontaneous miscarriage, divided into non-pregnant and pregnant women with previous spontaneous miscarriage. Second, the control group consisting of 35 healthy females who never had a miscarriage. The effects of different factors on the study parameters Dependent on the questionnaire form, seven parameters were used to classify the specimens according to the age, body-mass index (kg/m2), menopausal status, education level, number of miscarriages, number of children, and presence of other diseases. The findings of this study showed significant differences between study groups .The age (mean ± standard deviation) value of the spontaneous miscarriage group was 31.03 ± 7.71 while that for pregnancy group was 26.18 ± 4.35 years. According to a study by Gracia et al. [23], the age is considered as an independent risk factor for recurrent miscarriages. Women between the ages of 25 and 30 years had the lowest risk of spontaneous abortion, while the highest risk with an extreme level. Another study by Shawky et al. [24] reported that the number of miscarriage women at the age higher than 30 or the end of the 30s was the double compared to women without miscarriages. In addition, the present study found no significant differences in the parameters of body mass index, education level, menopausal status, number of miscarriage, and presence of other diseases (Table-2). Table 2General parameters of the study according to miscarriage status: spontaneous miscarriage group, pregnancy group (had a history of miscarriage) and control (never had a miscarriage). Total no. Control Spontaneous Miscarriage Pregnancy P-Value‡ Age (years) Mean ± SD Age 80 33.17 ± 7.78 31.03 ± 7.71 26.18 ± 4 .35 0.027 * Body-mass Index(kg/m2) (Mean ± SD) index(kg/m2) 80 28.08 ± 5.81 26.97 ± 4.02 25.12 ± 2.35 0.191 N.S Education level N (% of total) Middle school lower 40 16 (20.0%) 18 (22.5%) 6 (7.5%) 0.732 N.S High school 27 14 (17.5 %) 9(11.3%) 4 (5.0%) Akram and Kandala Iraqi Journal of Science, 2021, Vol. 62, No. 1, pp: 83-95 87 University 13 5 (6.3 %) 7 (8.8%) 1 (1.3%) Menopausal status N (% of total) Premenopausal 74 32 (40.0%) 31 (38.8%) 11 (13.8%) 0.596 N.S Postmenopausal 6 3 (3.8%) 3 (3.8%) 0 (0.0%) Number of children N (% of total) 1 or no 31 6 (7.5%) 18 (22.5%) 7 (8.8%) 0.003 ** 2 13 5 (6.3%) 7 (8.8%) 1 (1.3%) 3 or more 36 24 (30.0%) 9 (11.3%) 3 (3.8) No. of miscarriage N (% of total) 2 or less 29 0 23 (51.1%) 6 (13.3%) 0.724 N.S 3 10 0 7 (15.6%) 3 (6.7%) 4 or more 6 0 4 (8.9%) 2(4.4%)


Introduction
Miscarriage refers to pregnancy loss at less than 20 weeks of gestation; it is the most common complication of pregnancy. Several etiologies factors such as anatomic, genetic, endocrine, hematological and immunological factors have been reported to associate with this destructive complication of pregnancy [1,2]. Since the word "abortion" is associated with elective termination, the term "miscarriage" is synonymous and often used with patients [3]. First-trimester miscarriage refers to the early stage in the course of a miscarriage when intact gestational sacs are still present within the uterine cavity, blighted ovum; missed, delayed, silent miscarriage, or early fetal demise. The diagnosis of early fetal demise is based either on the absence of cardiac activity in a visible embryo or on the absence of an embryo within a gestational sac [4]. Second-trimester pregnancy loss is defined as pregnancy loss after the 14th week and before the 24th week of gestation. However, it has been associated with infection, cervical insufficiency, uterine malformations, gene polymorphisms, fetal and placental anomalies, and genetic and acquired thrombophilia [5]. Recurrent spontaneous miscarriage (RSM) is described as the occurrence of 3 or more consecutive spontaneous miscarriages, in a series of conditions that lead to or could terminate in reproductive failure [6].
Recurrent spontaneous miscarriage is multifactorial, so that approximately half of all cases will remain unexplained. At present, there exist a small number of accepted etiologies for RSM. These include uncontrolled diabetes mellitus, untreated hypothyroidism, parental chromosomal abnormalities, anti-phospholipid antibody syndrome (APS), and certain uterine anatomic abnormalities. Other probable or possible etiologies include heritable and/or acquired thrombophilia, immunologic abnormalities, additional endocrine disorders, environmental factors, and infection [7,8]. The correlation between the adverse outcomes of pregnancy and host infections was poorly examined, whereas intrauterine infections that occur in early and late pregnancy leading to a spontaneous miscarriage and spontaneous preterm birth were recorded [9]. Since the main route of placenta and fetus infection is from the vagina and cervix, most studies have attempted to find a link between miscarriage and abnormal bacterial flora in the lower genital tract [10]. However, bacterial vaginosis was reported to be causative for second-trimester miscarriage or premature delivery in the first trimester of pregnancy [11], although bacterial vaginosis-related bacteria and their toxins can cross the placenta and cause fetal complications [12].
Atopobium vaginaean bacteria are anaerobic, Gram-positive, non-mobile, sporogenous and noncapsulated, measuring approximately 0.6-0.9 μm and forming small clear colonies similar to small pinheads in Columbia blood agar cultures at 37°C [13]. In addition to the highlighted position in BV, A. vaginae infection and a higher risk of premature birth was associated [14]. Leptotrichia species are fastidious gram-negative, non-motile, long, fusiform rods, non-spore forming, anaerobic bacteria that exist as part of microorganisms of the human oral cavity and female genitalia [15]. A Leptotrichia sp. was first reported in 2002, when Shukla et al. [16] described L. amnionii as a novel causative species of infection in the female urogenital tract. Also, L. amnionii was isolated from the cervix of a patient with a premature rupture of the fetus membranes [17]. PCR analysis has been useful in detecting infections in patients with premature labour, intact membranes, and intra-amniotic infections compared to normal microbial cultures [18]. However, epidemiological data on the potential association between cervical microbiota and spontaneous miscarriage has been rarely reported. The objective of this study was to determine the cervical bacteria in different miscarriage samples using qPCR technique and investigate the correlation between spontaneous miscarriage and cervical bacteria in a sample of Iraqi women.

Materials and Methods The Subjects
All samples required in this study were collected from women (age ranged 18-51 years) attended as outpatients of gynecology department of the Medical City Teaching Hospital. Patient's samples were selected according to questionnaire information, history of spontaneous abortion, and number of abortions (and other categories). The study included 80 females, 45 suffering a recurrent spontaneous miscarriage and 35 normal). This study was approved by the Ethics and Research Committee of the hospital under the supervision of the consultant, while approval for the sampling was obtained from patients and controls.

Specimen collection
Two types of samples were collected from women; first, 80 cervical swabs from the endocervical region after inserting a sterile speculum into the vagina. Second, blood samples (5 of venous blood) which were then transferred into polyethylene tubes, allowed to clot, and centrifuged at 3000 rpm for 10 minutes. The obtained clear serum was frozen at -20 ºC until used for further immunological assays.

Immunochromatography detection of antibodies
In order to detect some microorganisms that maybe related to miscarriage in women, TORCH is a rapid test kit used in this study for the qualitative detection of TOX-IgM, RU-IgM, CMV-IgM, HSV-2-IgM, and HSV-1-IgM antibodies in human serum. The procedure followed the manufacturer's instructions (TORCH rapid test kit, Weifang Kanghua Biotech\China) [19]

Determination of Anticardiolipin IgM and Antiphospholipid IgM antibodies
The detection of antiphospholipid (APL) IgM and anticardiolipidin(ACL) IgM antibodies in the serum by was conducted using ELISA Kit, following the manufacturer's instructions (Antiphospholipid and Anticardiolipin antibodies ELISA Kit, Cortez \ USA) [20,21].

Extraction of DNA
Total DNA (genomic and bacterial DNA) was isolated from cervical swab samples for molecular studies following the standard protocol for extraction. Genomic DNA isolation was achieved by using a DNA-sorb-A nucleic acid extraction kit. Nanodrop instrument (Nas-99/China) was used to determine DNA concentration and purity. The integrity of DNA was estimated by agarose gel electrophoresis.

Real -Time Polymerase Chain Reaction For the qualitative detection of Leptotrichia amnionii and Atopobium vaginae
Real Time-for L. amnionii and A. Vaginae were was conducted by using 16S rRNA primers (Table-1). The reaction mixture consisted of 5μl DNA template, 0.8μl of each primer, 1.25µl CXR dye, 12.5μl of Bright Green Express 1X qPCR Master mix, and 4.65µl Nuclease-free water, in a total volume of 25μl. The template DNA was amplified for 40 cycles of denaturation for 1 minute at 95°C, annealing of primers at 63°C for A. vaginae and 64°C for L. amnionii for 1 minute, and extension at 72°C for 1 minute. Fluorescent data were acquired during each extension phase [22].

Statistical Analysis
The SPSS software (V 20.0) was utilized to estimate the effects of different factors on the study parameters. The correlation amongst the parameters was estimated using Pearson chi-squared (R) to test for significant differences at p< 0.05. Chi-squared and ANOVA tests were used to assess the differences in the categorical and continuous variables, respectively, among miscarriage patients.

Results and Discussion
A total of 80 specimens of endocervical cytobrush and blood were collected from both patients and control groups by a specialist physician at the gynecology department of the Medical City Teaching Hospital. These samples were divided into two groups. First, the patients group consisting of 45 women suffering a spontaneous miscarriage, divided into non-pregnant and pregnant women with previous spontaneous miscarriage. Second, the control group consisting of 35 healthy females who never had a miscarriage.

The effects of different factors on the study parameters
Dependent on the questionnaire form, seven parameters were used to classify the specimens according to the age, body-mass index (kg/m2), menopausal status, education level, number of miscarriages, number of children, and presence of other diseases. The findings of this study showed significant differences between study groups .The age (mean ± standard deviation) value of the spontaneous miscarriage group was 31.03 ± 7.71 while that for pregnancy group was 26.18 ± 4.35 years. According to a study by Gracia et al. [23], the age is considered as an independent risk factor for recurrent miscarriages. Women between the ages of 25 and 30 years had the lowest risk of spontaneous abortion, while the highest risk with an extreme level. Another study by Shawky et al. [24] reported that the number of miscarriage women at the age higher than 30 or the end of the 30s was the double compared to women without miscarriages.
In addition, the present study found no significant differences in the parameters of body mass index, education level, menopausal status, number of miscarriage, and presence of other diseases (Table-2). Non-significant (NS),*significant <0.05

Detection of TORCH by Immunochromatography Test
The findings showed that all the 45 specimens of females with recurrent spontaneous miscarriage were negative for TORCH test (TOX-IgM, RU-IgM, CMV-IgM, HSV-2-IgM, and HSV-1-IgM). These findings confirmed that these cases did not included congenital and perinatal infections, as previously indicated by Suryawanshi and co-workers [25].
In spite of that, other studies revealed the effects of pathogens including toxoplasmosis, rubella, cytomegalovirus and herpes simplex. Thus, the role of TORCH complex in determining abortion remains controversial [26], as little information is available about the impact of TORCH confections on the outcome of pregnancy and the causes of miscarriage [27].

Determination of Anticardiolipin IgM and Antiphospholipid IgM antibodies
Out of 45 miscarriage females included, 34 were with spontaneous miscarriage and 11 were under pregnancy. All the specimens were in the normal range ACL (<7 U/ml) APL (<12U/ml) IgM antibodies, indicative that both antibody types do not have a role in miscarriage cases. A previous study [28] indicated that females with antiphospholipid syndrome also have a higher risk of miscarriages, with recurrent miscarriages in some cases becoming an indicator for antiphospholipid testing. Therefore, the possibility of miscarriage due to these factors was excluded in this.

Molecular Detection Extraction of DNA
Total DNA (genomic and bacterial DNA) was extracted from cervical swab specimens for molecular studies. The results showed that the purity ranged 1.7-2.0 and the concentration ranged 20-50 μg/ μL (Figure-1).

Detection of A.vaginae Using Real -Time PCR Technique
According to the standard curve of primer efficiency, the positive result of A.vaginae is ranging from 13 to 27 Ct (cycle threshold), as shown in Figure-2. The results showed that out of a total of 80 specimens, including 34 of spontaneous miscarriages, 11 pregnancies, and 35 controls, only 6, 1, and 5 specimens, respectively, were positive for A. vaginae. The green fluorophore channel was used to detect the 16S rRNA gene of A. vaginae in the samples (Figure-3), and the melting curves are shown in (Figure-4). The findings agree with a study by Harper et al. [29] who noticed no evidence of a synergistic interaction for the coexistence of vaginosis bacteria and periodontal disease on preterm birth. While the study of Knoester et al. [30] suggested an association between maternal bacteremia with A. vaginae and fetal death following Chorionic Villus Sampling. Another local study by Mahdey et al. [31] specified a high risk of abortion with the infection by Megasphera and Atobopium. The study applied qPCR for 16S rRNA gene of bacterial vaginosis as a target with specific and sensitive diagnosis in 100 swabs of Iraqi women with vaginitis.   However, the products of RT-PCR were analyzed by gel electrophoresis to detect the product length for 16S rRNA gene. The results revealed that the molecular size of A. vaginae after staining with ethidium bromide and visualization under UV light was of 558 bp, as shown in (Figure -5). Also, the product length was used as an internal control for the gene [32].

Qualitative Detection of L.amnionii Using Real Time -PCR
The positive result of L.amnionii has a range of 14-26, which is represented in Ct according to the standard curve of primer efficiency, as shown in (Figure -6).
The results of RT-qPCR detection showed that, out of 34 specimens of spontaneous miscarriage, 11 pregnancies, and 35 controls, only 11, 4, and 2 specimens, respectively, were positive for L. amnionii.  (Figure -9). The findings agree with the study of Shukla et al. [16] who reported that a novel bacterium was isolated from the amniotic fluid of women who experienced intrauterine fetal death during the second trimester of pregnancy.

Akram and Kandala
Iraqi Journal of Science, 2021, Vol. 62, No. 1, pp: 83-95 92 However, the products of RT-PCR were analyzed by gel electrophoresis to detect the product length for 16S rRNA gene of L.amnionii after staining with ethidium bromide and visualization under UV light, which had a molecular size of 229bp as shown in (Figure -10). Also, the product length was used as an internal control for the gene [32].

Association between A.vaginae, L.amnionii, and Spontaneous Miscarriage
The results of this study showed no significant associations (P= 0.778) between infections with A.vaginae and spontaneous miscarriage, according to the results of RT-PCR for 16S rRNA gene that were positive in 6/34 (17.6 %) of the spontaneous miscarriage group and 1/11 (9.1%) of the pregnancy group, compared with 5/35(14.3%) in the control group. These results disagree with other studies which showed that the infection with A.vaginae was strongly associated with spontaneous miscarriage [33,34].
The results in Table -3 show no significant association between A. vaginae and spontaneous miscarriage and pregnancy groups compared with the control group (p= 0.778). The adjusted ORs of A. vaginae for spontaneous miscarriage and pregnancy were 1.23 (CI 95%, 0.416 -3.67) and 0.636 (CI 95%, 0.083 -4.88) with p-value of 0.703 and 0.655, respectively. These results do not support the assumption that spontaneous miscarriage also associates with the involvement of A. vaginae. Infection with A. vagina was reported to possibly cause maternal sepsis and spontaneous miscarriage [30].
Meanwhile, the results of L. amnionii show significant association with spontaneous miscarriage and pregnancy groups compared with the control group (p= 0.011). The adjusted ORs of L. anmionii for spontaneous miscarriage and pregnancy were 7.89 (CI 95%, 1.60 -39.00) and 9.43 (CI 95%, 1.43 -61.9), showing highly significant association with p-value of 0.005 and 0.008, respectively, compared with the control group. These results support the claim that a previous Norwegian case study of L. amnionii was the first to differentiate patients with chorioamnionitis L. amnionii from a renal abscess in spontaneous miscarriage [17]. Cumulatively, these results suggest a prospective look into the association between the microbiota, including L. amnionii, found in the cervix with spontaneous miscarriage.

Control Spontaneous miscarriage Pregnancy
In conclusion, fetal loss is considered high in women in the age range of the 30s. This is due to that recurrent miscarriage and other complications in pregnant women might share underlying causes, which could be biological conditions. Infection with L. amnionii showed a strong association with spontaneous miscarriage and pregnancy groups, which found highly significant differences compared with the results of A. vaginae in the two groups.