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New biopsy after antibiotic treatment: effect on outcomes of assisted reproduction in patients with infertility and chronic endometritis

  • Author Footnotes
    # Contributed equally.
    Wen-juan Liu
    Footnotes
    # Contributed equally.
    Affiliations
    The First Affiliated Hospital of Jinan University, 613 Huangpu Avenue West, Tianhe District, Guangzhou Guangdong Province 510630, China

    Department of Reproductive Medical Center, Guangdong Women and Children Hospital, No. 521 Xingnan Road, Guangzhou Guangdong Province 511400, China
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  • Author Footnotes
    # Contributed equally.
    Ju Huang
    Footnotes
    # Contributed equally.
    Affiliations
    The First Affiliated Hospital of Jinan University, 613 Huangpu Avenue West, Tianhe District, Guangzhou Guangdong Province 510630, China

    Department of Reproductive Medical Center, Guangdong Women and Children Hospital, No. 521 Xingnan Road, Guangzhou Guangdong Province 511400, China
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  • Li Sun
    Affiliations
    Department of Reproductive Medical Center, Guangdong Women and Children Hospital, No. 521 Xingnan Road, Guangzhou Guangdong Province 511400, China
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  • Li Huang
    Affiliations
    Department of Reproductive Medical Center, Guangdong Women and Children Hospital, No. 521 Xingnan Road, Guangzhou Guangdong Province 511400, China
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  • Qian-yu Zhang
    Affiliations
    Department of Reproductive Medical Center, Guangdong Women and Children Hospital, No. 521 Xingnan Road, Guangzhou Guangdong Province 511400, China
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  • Ying-qi Nong
    Affiliations
    Department of Reproductive Medical Center, Guangdong Women and Children Hospital, No. 521 Xingnan Road, Guangzhou Guangdong Province 511400, China
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  • Jia-hui Wei
    Affiliations
    Department of Reproductive Medical Center, Guangdong Women and Children Hospital, No. 521 Xingnan Road, Guangzhou Guangdong Province 511400, China
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  • Kun-he Wu
    Affiliations
    Department of Pathology, Guangdong Women and Children Hospital, No. 521 Xingnan Road, Guangzhou Guangdong Province 511400, China
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  • Feng-hua Liu
    Correspondence
    Corresponding author.
    Affiliations
    The First Affiliated Hospital of Jinan University, 613 Huangpu Avenue West, Tianhe District, Guangzhou Guangdong Province 510630, China

    Department of Reproductive Medical Center, Guangdong Women and Children Hospital, No. 521 Xingnan Road, Guangzhou Guangdong Province 511400, China
    Search for articles by this author
  • Author Footnotes
    # Contributed equally.
Open AccessPublished:August 06, 2022DOI:https://doi.org/10.1016/j.rbmo.2022.07.020

      Highlights

      • Doxycycline-treated women with CE and infertility were re-examined
      • The endometrial re-examination did not affect pregnancy outcomes
      • The 1st doxycycline treatment cycle improved outcomes in women with CD138+/HPF≥5
      • Multiple endometrial biopsies are not required for some CE patients

      Abstract

      Research question

      What is the effect of chronic endometritis on patients with infertility, the necessity of endometrial re-examination and the effect of improving chronic endometritis after one cycle of antibiotic treatment on pregnancy outcomes?

      Design

      Infertile patients (n = 4003) who underwent IVF and intracytoplasmic sperm injection treatment were included. Pregnancy outcomes of groups positive for chronic endometritis were compared with groups that were negative (group 1). Patients that were positive were divided into the chronic endometritis new biopsy group (group 2) and chronic endometritis non-re-examination group (group 3). After doxycycline treatment and re-examination, the chronic endometritis new biopsy group was divided into improved chronic endometritis group (ICE) and not-improved chronic endometritis group (NICE), and their general indicators and reproductive outcomes were compared.

      Results

      No significant difference was observed in embryo implantation, early or late pregnancy loss, ectopic pregnancy, clinical pregnancy and live birth rates between groups 2 and 3. The clinical pregnancy and live birth rates in the NICE group were significantly lower than those in the ICE group (P = 0.008 and P = 0.001, respectively). After controlling for potential confounding factors, age, average number of high-quality embryos, endometrial thickness on the day of embryo transfer and number and type of embryo transfer were factors associated with live birth rates.

      Conclusions

      Endometrial re-examination of women with chronic endometritis treated with doxycycline had no effect on pregnancy outcomes. The first cycle of doxycycline treatment could effectively improve reproductive outcomes of women with five or more CD138+ cells/high-power field.

      KEYWORDS

      Introduction

      Chronic endometritis is persistent inflammation in the endometrium and may be induced by various pathogens (
      • Greenwood S.M.
      • Moran J.J.
      Chronic endometritis: morphologic and clinical observations.
      ). Chronic endometritis is easily overlooked, as most patients do not show obvious symptoms and signs or even mild symptoms, such as abnormal uterine bleeding, pelvic pain, dyspareunia and leucorrhoea for extended periods (
      • Cicinelli E.
      • Trojano G.
      • Mastromauro M.
      • Vimercati A.
      • Marinaccio M.
      • Mitola P.C.
      • Resta L.
      • de Ziegler D.
      Higher prevalence of chronic endometritis in women with endometriosis: a possible etiopathogenetic link.
      ). The prevalence of chronic endometritis in the general population is unclear and ranges from 0.8% to 27.1% (
      • Johnston-MacAnanny E.B.
      • Hartnett J.
      • Engmann L.L.
      • Nulsen J.C.
      • Sanders M.M.
      • Benadiva C.A.
      Chronic endometritis is a frequent finding in women with recurrent implantation failure after in vitro fertilization.
      ). In patients with infertility, however, the prevalence ranges from 3% to 44% (
      • Cicinelli E.
      • Resta L.
      • Nicoletti R.
      • Zappimbulso V.
      • Tartagni M.
      • Saliani N.
      Endometrial micropolyps at fluid hysteroscopy suggest the existence of chronic endometritis.
      ;
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      • Fatemi H.M.
      • Bourgain C.
      • Sie-Go D.M.
      • Eijkemans R.J.
      • Fauser B.C.
      • Devroey P.
      • Broekmans F.J.
      The impact of chronic endometritis on reproductive outcome.
      ;
      • Kasius J.C.
      • Broekmans F.J.M.
      • Sie-Go D.M.D.S.
      • Bourgain C.
      • Eijkemans M.J.C.
      • Fauser B.C.
      • Devroey P.
      • Fatemi H.M.
      The reliability of the histological diagnosis of endometritis in asymptomatic IVF cases: a multicenter observer study.
      ), even reaching 57% (
      • Cicinelli E.
      • Matteo M.
      • Tinelli R.
      • Lepera A.
      • Alfonso R.
      • Indraccolo U.
      • Marrocchella S.
      • Greco P.
      • Resta L.
      Prevalence of chronic endometritis in repeated unexplained implantation failure and the IVF success rate after antibiotic therapy.
      ) in those with unexplained recurrent miscarriage or repeated implantation failures.
      Currently, methods for diagnosing chronic endometritis include endometrial immunohistochemistry (IHC), hysteroscopy and microbiology. Among them, IHC and hysteroscopy are highly subjective and not recognized by standards established by consensus. Pathological examination shows that plasma cell infiltration in the endometrial stroma is the gold standard for diagnosing chronic endometritis. Traditionally, plasma cells have been identified in haematoxylin and eosin-stained specimens. The identification of plasma cells in haematoxylin and eosin sections requires experience. Immunohistochemistry analysis of syndecan-1 (CD138), a transmembrane-type heparan sulphate proteoglycan on the plasma cell surface and keratinocytes, has been introduced recently to improve the sensitivity and accuracy of plasma cell identification and help in the diagnosis of chronic endometritis (
      • Bayer-Garner I.B.
      • Nickell J.A.
      • Korourian S.
      Routine syndecan–1 immunohistochemistry aids in the diagnosis of chronic endometritis.
      ;
      • Kitaya K.
      • Yasuo T.
      Inter–observer and intra–observer variability in immunohistochemical detection of endometrial stromal plasmacytes in chronic endometritis.
      ).
      Criteria for diagnosing chronic endometritis vary, i.e. the total number of plasma cells on each slide, the number of plasma cells per 1 or 10 high-power fields (HPF) and the average number of plasma cells under multiple HPF. Moreover, the threshold for the number of plasma cells used for the diagnosis varies in different studies. Plasma cell density has been considered a standard for diagnosis (
      • Liu Y.
      • Chen X.
      • Huang J.
      • Wang C.C.
      • Yu M.Y.
      • Laird S.
      • Li T.C.
      Comparison of the prevalence of chronic endometritis as determined by means of different diagnostic methods in women with and without reproductive failure.
      ), but no standard has been agreed for diagnosing chronic endometritis using CD138 IHC (
      • Margulies S.L.
      • Dhingra I.
      • Flores V.
      • Hecht J.L.
      • Fadare O.
      • Pal L.
      • Parkash V.
      The diagnostic criteria for chronic endometritis: A survey of pathologists.
      ;
      • Li Y.
      • Xu S.
      • Yu S.
      • Huang C.
      • Lin S.
      • Chen W.
      • Mo M.
      • Lian R.
      • Diao L.
      • Ding L.
      • Zeng Y.
      Diagnosis of chronic endometritis: How many CD138(+) cells/HPF in endometrial stroma affect pregnancy outcome of infertile women?.
      ). In addition, the time needed for endometrial biopsy and the size of biopsy specimens may affect the accuracy of IHC staining. Specimens collected in the proliferative phase show a higher possibility of plasma cell infiltration than those collected in the secretory phase (
      • Song D.
      • Feng X.
      • Zhang Q.
      • Xia E.
      • Xiao Y.
      • Xie W.
      • Li T.C.
      Prevalence and confounders of chronic endometritis in premenopausal women with abnormal bleeding or reproductive failure.
      ).
      Microbial infection of the uterine cavity is considered the primary cause of chronic endometritis (
      • Cicinelli E.
      • De Ziegler D.
      • Nicoletti R.
      • Colafiglio G.
      • Saliani N.
      • Resta L.
      • Rizzi D.
      • De Vito D.
      Chronic endometritis: correlation among hysteroscopic, histologic, and bacteriologic findings in a prospective trial with 2190 consecutive office hysteroscopies.
      ;
      • Moreno I.
      • Simon C.
      Relevance of assessing the uterine microbiota in infertility.
      ). Endometrial tissue culture can identify the source pathogen of chronic endometritis, and a drug sensitivity test provides medication guidance for subsequent treatment. Not all microorganisms, however, can be examined using ordinary culture methods, and pathogen culture is time-consuming (
      • Moreno I.
      • Cicinelli E.
      • Garcia-Grau I.
      • Gonzalez-Monfort M.
      • Bau D.
      • Vilella F.
      • De Ziegler D.
      • Resta L.
      • Valbuena D.
      • Simon C.
      The diagnosis of chronic endometritis in infertile asymptomatic women: a comparative study of histology, microbial cultures, hysteroscopy, and molecular microbiology.
      ). In addition, endometrial biopsy is easily contaminated by vaginal secretions, eventually leading to inaccurate culture results. Therefore, in most cases, chronic endometritis is treated with oral antibiotics. The treatment, however, is mostly empirical, whereas improper treatment can lead to recurrence of chronic endometritis or drug resistance. At present, the effect of chronic endometritis on patients with infertility remains controversial.
      Therefore, the aim of the present study was to investigate the feasibility of antibiotic treatment alone for patients with infertility in conjunction with chronic endometritis without re-examination using retrospective cohort data. The necessity of re-examination after antibiotic treatment was assessed, and the effects of histologically confirmed chronic endometritis on pregnancy outcomes was analysed in patients with infertility who underwent IVF.

      Materials and methods

      Participants

      A total of 5365 patients with infertility underwent hysteroscopy at the Reproductive Medical Centre, Guangdong Women and Children Hospital, between March 2018 and January 2020. Endometrial tissue biopsy was carried out by CD138 IHC analysis 6 months before IVF and intracytoplasmic sperm injection (ICSI) treatment. Patients who did not receive antibiotic treatment before hysteroscopy, those aged between 20 and 45 years and those who had two or more embryos available, including at least one high-quality embryo (according to Racowsky's cleavage-stage embryo grade (
      • Racowsky C.
      • Stern J.E.
      • Gibbons W.E.
      • Behr B.
      • Pomeroy K.O.
      • Biggers J.D.
      National collection of embryo morphology data into Society for Assisted Reproductive Technology Clinic Outcomes Reporting System: associations among day 3 cell number, fragmentation and blastomere asymmetry, and live birth rate.
      ) and Gardner's blastocyst grade (
      • Gardner D.K.
      • Lane M.
      • Stevens J.
      • Schlenker T.
      • Schoolcraft W.B.
      Blastocyst score affects implantation and pregnancy outcome: towards a single blastocyst transfer.
      ), were included in the study. Patients with uterine adhesion, submucosal myoma, hydrosalpinx, adenomyosis, endometriosis stage III and above, uterine malformations, recurrent implantation failure and recurrent pregnancy loss, and preimplantation genetic testing cycles were excluded. Finally, a total of 4003 embryo transfer cycles were analysed retrospectively. The study flowchart is presented in Figure 1. The present study was approved by the Ethics Committee of Guangdong Women and Children Hospital (approval number: 202101341,12/21/2021), and was carried out in accordance with the principles of the Declaration of Helsinki.
      Figure 1
      Figure 1Study flowchart and grouping. Chronic endometritis-negative group: fewer than five CD138+ cells/high-power field (HPF); chronic endometritis-positive group: five or more CD138+ cells/HPF.

      Diagnostic criteria and treatment of chronic endometritis

      All patients underwent hysteroscopy in the proliferative phase (3–7 days after menstruation) or when the amount of bleeding was low in the case of abnormal vaginal bleeding. The uterine cavity was examined thoroughly. Endometrial biopsy samples were collected using biopsy forceps during hysteroscopy. All endometrial biopsy samples were sent to the same laboratory and interpreted by experienced pathologists specializing in endometrial pathology. The presence of plasma cells in the endometrial stroma examined by CD138 IHC staining was considered the histological diagnostic marker. After scanning at lower magnification, the number of plasma cells per microscopic HPF (400) (CD138/HPF) was counted to evaluate the largest number of plasma cells. Pathological results combined with IHC analysis were used as the criteria for the diagnosis of chronic endometritis (
      • Bouet P.E.
      • El Hachem H.H.
      • Monceau E.
      • Gariépy G.
      • Kadoch I.J.
      • Sylvestre C.
      Chronic endometritis in women with recurrent pregnancy loss and recurrent implantation failure: prevalence and role of office hysteroscopy and immunohistochemistry in diagnosis.
      ): typical infiltration of endometrial stroma by plasma cells and five or more CD138-positive plasma cells per 10 random stromal areas at 400 × magnification.
      All patients with five or more CD138+ cells/HPF received at least one course of oral antibiotic treatment. For the first treatment course, doxycycline (100 mg orally twice a day for 14 days) was administered. If five or more CD138+ cells/HPF were observed in subsequent endometrial biopsy samples, levofloxacin lactate (200 mg orally twice a day) plus metronidazole (500 mg orally three times a day) was administered for 14 days. If five or more CD138+ cells/HPF was observed again in the endometrium after two courses of antibiotic treatment, persistent chronic endometritis infection was diagnosed. Levofloxacin lactate (200 mg orally twice a day) plus metronidazole (500 mg orally three times a day) for another 14 days was prescribed as the third treatment course (Figure 1).

      Grouping of patients with chronic endometritis

      On the basis of inclusion and exclusion criteria, infertile patients were divided into chronic endometritis negative (Group 1) and chronic endometritis positive groups. Chronic endometritis positive groups were all treated with doxycycline and were further divided into a new biopsy group (group 2) and non-re-examination group (group 3). The effect of endometrial new biopsies on pregnancy outcomes in patients with chronic endometritis was examined.
      Next, the effect of chronic endometritis improvement after one cycle of antibiotic treatment on pregnancy outcomes was examined. Data from group 2, which was divided into the improved chronic endometritis group (ICE) and not-improved chronic endometritis group (NICE), were retrospectively analysed. The NICE group was treated with levofloxacin or metronidazole. The flowchart of grouping is presented in Figure 1.

      Re-examination by new endometrial biopsy

      Patients with five or more CD138+ cells/HPF underwent endometrial biopsy for histological re-evaluation of the endometrium after antibiotic treatment in the proliferative phase. The medical staff confirmed the depth and direction of the patients’ uterine cavity, placed a disposable endometrial collector (SAP-1) (Beijing Saipu Jiuzhou Technology Development Co., Ltd., China) at their fundus uteri, fully opened the collector and rotated it clockwise three to five times so that the inner membrane was fully stimulated. The inner membrane tissues were harvested for pathological examination and IHC analysis for CD138. After surgery, patients were administered routine infection prevention and control treatment for 3 days.

      Ovarian stimulation protocols

      Two protocols, including a gonadotrophin-releasing hormone (GnRH) antagonist protocol and GnRH agonist long protocol, were selected for ovarian stimulation in infertile women according to their characteristics. For the GnRH agonist protocol, 1.0 mg of long-acting GnRH-a (Diphereline) (Ipsen, Paris-Saclay, France) was injected during the middle of the luteal phase of the menstrual cycle in the participants before retrieving follicles. For the two protocols, stimulation was initiated by administering 100–300 IU/day of gonadotrophin: GONAL-f (Merck Serono Pharmaceuticals, Darmstadt, Germany) or Puregon (Merck Sharp & Dohme, Whitehouse Station, NJ, USA) to participants based on the basal FSH level, anti-Müllerian hormone (AMH) level, antral follicular count and maternal age on days 2–5 of the menstrual cycle. Gonadotrophin dosage was adjusted according to serum hormone levels and follicle measurements. Next, ovulation was induced by adding an antagonist (Ganirelix Acetate) (Merck Sharp & Dohme, Whitehouse Station, NJ, USA) until the day of HCG administration.

      IVF and fresh embryo transfer strategy

      The details of the IVF/ICSI procedure, embryo culture and evaluation, vitrification and warming of embryos, and frozen embryo transfer (FET) have been described previously (
      • Liu W.J.
      • Nong Y.Q.
      • Ruan J.X.
      • Chen Y.
      • Fan L.
      • Huang Q.W.
      • Liu F.H.
      Impact of endometrial thickness during menstruation and endometrial scratching on the pregnancy in frozen–thawed embryo transfer.
      ;
      • Zhou R.
      • Zhang X.
      • Dong M.
      • Huang L.
      • Zhu X.
      • Wang S.
      • Liu F.
      Association between endogenous LH level prior to progesterone administration and live birth rate in artificial frozen–thawed blastocyst transfer cycles of ovulatory women.
      ). IVF or ICSI was carried out depending on the semen quality. Normal fertilization was assessed 16–18 h after insemination/injection, and the embryos were subsequently cultured until day 3 or day 5/6. They were subjected to embryo transfer or whole embryo vitrification based on the progesterone concentration as well as the risk of ovarian hyperstimulation syndrome, hydrosalpinx, and endometrium thickness. For embryo transfer, from the day of egg retrieval, patients received 10 mg of dydrogesterone twice a day and 90 mg of crinone (Fleet Laboratories Limited, Watford, Hertfordshire, UK) once a day by vaginal suppository or 40 mg of progesterone injected intramuscularly once a day for luteal support. Embryos were transferred under ultrasound guidance using a Cook catheter (Curved Embryo Transfer Catheter) (Cook Medical, Bloomington, USA). One or two warmed embryos were transferred depending on the age of the women, embryo quality and cycle rank.

      Frozen–thawed embryo transfer strategy (using vitrified embryos)

      The FET cycles were carried out within 6 months after antibiotic treatment. Endometrial preparation was carried out as previously described (
      • Zhou R.
      • Zhang X.
      • Dong M.
      • Huang L.
      • Zhu X.
      • Wang S.
      • Liu F.
      Association between endogenous LH level prior to progesterone administration and live birth rate in artificial frozen–thawed blastocyst transfer cycles of ovulatory women.
      ). The natural cycle was used for patients with regular menstrual cycles, and a hormone therapy cycle or stimulation cycle was used for patients with irregular menstrual cycles. The luteal support programme and embryo transfer strategy were conducted in the same manner as those during the embryo transfer cycle.

      Assessments

      The implantation rate, clinical pregnancy rate, early pregnancy loss rate, late pregnancy loss rate, ectopic pregnancy rate and live birth rate were retrospectively evaluated. The implantation rate was defined as the percentage of embryos implanted successfully relative to the total number of embryos transferred. Clinical pregnancy was defined as the appearance of an intrauterine gestational sac with positive cardiac movement as documented via transvaginal ultrasonography. Live birth was defined as the birth of at least one newborn after 24 weeks of gestation. Early pregnancy loss was defined as a spontaneous clinical pregnancy loss during the first trimester.

      Statistical analysis

      The Statistical Package for Social Sciences (SPSS) version 22.0 was used for all statistical analyses. Data with normal distribution are presented as mean ± SD. When the variances were equal, one-way analysis of variance was used for analysis. Data that did not follow normal distribution were presented as median (interquartile range) M (P25-P75), and the Mann–Whitney U test was used for analysis. The differences in variables among groups were analysed using the chi-squared test or Fisher's exact test and a one-way analysis of variance. Multivariable logistic regression was carried out to explore the effect of chronic endometritis on the live birth for cleavage-stage embryo transfer and blastocyst transfer, after controlling for potential confounding factors. These included maternal age, average number of high-quality embryos, embryo transfer or FET, types and number of transferred embryos and endometrium thickness on the day of embryo transfer. Results are reported as adjusted odds ratios with 95% confidence intervals. P < 0.05 was considered statistically significant.

      Results

      Infection, treatment and cure rate of antibiotic treatment in women with chronic endometritis, and general characteristics of the study population
      Of the 5365 women who underwent endometrial biopsy after routine hysteroscopy, a total of 4003 women were included in the analysis (Figure 1). Among the 4003 women, 2742 (68.50%) in the chronic endometritis-negative group (group 1) were determined to have fewer than five CD138+ cells/HPF by histological examination at first evaluation, and 1261 (31.50%) were determined to have five or more CD138+ cells/HPF. After treatment with the first cycle of doxycycline, the chronic endometritis-positive groups were divided into a new biopsy group (group 2, n = 927) and non-re-examination group (group 3, n = 334). All 927 women received the first cycle of oral antibiotic treatment and underwent endometrial biopsy, 733 (79.07%) were cured with fewer than five CD138+ cells/HPF, whereas 194 (20.93%) still had five or more CD138+ cells/HPF and were treated with levofloxacin/metronidazole.
      No differences were found among the three groups in age, years of infertility, infertility factors, body mass index, AMH levels, basic FSH levels, basic oestradiol levels, basic progesterone levels, gonadotrophin starting dose, total gonadotrophin dose, number of follicles obtained and normal fertilization rate. The average number of high-quality embryos in the chronic endometritis non-re-examination group was lower than that in the chronic endometritis re-examination group (P = 0.034) (Table 1).
      TABLE 1COMPARISON OF MATERNAL AND TREATMENT CHARACTERISTICS AMONG DIFFERENT CHRONIC ENDOMETRITIS GROUPS AND THE CHRONIC ENDOMETRITIS-NEGATIVE GROUP
      Chronic endometritis-negative groupChronic endometritis-positive groupF/χ2 valueP-value
      Re-examination groupNon-re-examination group
      Number of cycles2742927334
      Age32.78 ± 4.5932.54 ± 4.5032.82 ± 4.742.5900.075
      Years of infertility3.79 ± 2.973.65 ± 3.013.87± 3.401.4080.245
      Infertility causes1.8690.760
       Oviduct, % (n)48.18 (1321/2742)47.68 (442/927)51.20 (171/334)
      Male factors, % (n)26.88 (737/2742)27.83 (258/927)26.65 (89/334)
      Other, % (n)24.94 (684/2742)24.49 (227/927)22.15 (74/334)
      BMI, kg/m221.73 ± 3.0021.93 ± 2.8621.83 ± 2.760.1100.896
      AMH, ng/ml3.60 (1.87–6.39)3.59 (1.79–5.82)3.71 (1.72–6.35)0.680
      Basic FSH IU/l6.91 (5.84–8.08)6.67 (5.79–7.93)6.90 (5.85–8.09)0.121
      Basic oestradiol pg/ml39.10 (29.56–51.60)39.00 (29.51–50.79)39.90 (30.01–52.39)0.341
      Basic progesterone ng/ml0.25 (0.14–0.40)0.25 (0.15–0.38)0.24 (0.15–0.36)0.288
      Gonadotrophin starting dose, IU189.47 ± 67.14189.74 ± 67.84189.37 ±67.7551.3050.271
      Gonadotrophin duration11.29 ± 2.2810.90 ± 2.1811.04 ± 2.212.8390.059
      Total gonadotrophin dose, IU2339.98 ± 893.992218.91 ± 880.782313.64 ± 925.440.0860.918
      Follicles obtained, n15.03 ± 8.7014.97 ± 8.6713.88 ± 9.462.5410.079
      Normal fertilization rate, % (n)66.83 (25053/37488)67.70 (8570/12658)66.62 (2826/4242)3.6050.165
      Mean number of high-quality embryos6.68 ± 4.466.84 ±4.616.11 ± 4.55
      P = 0.034 compared with the chronic endometritis re-examination group. AFC, antral follicular count; AMH, anti-Mullerian hormone; BMI, body mass index.
      3.2320.040
      Data presented as mean ± SD, % (n) and median (interquartile range).
      a P = 0.034 compared with the chronic endometritis re-examination group.AFC, antral follicular count; AMH, anti-Mullerian hormone; BMI, body mass index.

      Comparison of pregnancy outcomes of different chronic endometritis groups with those of the chronic endometritis-negative group

      The percentage of embryo transfer or frozen embryo transfer in group 3 was higher than that in the other two groups (P < 0.001). The proportion of double cleavage-stage embryo transfer in group 3 was higher than that in group 1 and group 2, whereas the proportion of single blastocyst transfer (SBT) in group 3 was less than that in the other two groups (P < 0.001). No significant differences were found in endometrial thickness on the day of embryo transfer, embryo implantation rate, early pregnancy loss rate, late pregnancy loss rate, ectopic pregnancy rate, clinical pregnancy rate and live birth rate among the three groups (Table 2).
      TABLE 2COMPARISON OF THE PREGNANCY OUTCOMES AMONG DIFFERENT CHRONIC ENDOMETRITIS GROUPS AND THE CHRONIC ENDOMETRITIS-NEGATIVE GROUP
      CharacteristicsChronic endometritis -negative groupChronic endometritis-positive groupF/χ2 valueP-value
      Re-examination groupNon-re-examination group
      Cycles, n2742927334
      Embryo transer/FET, % (n)64.49 (1075/1667)55.80 (332/595)116.88 (180/154)
      P < 0.001 the embryo transfer/FET (%) compared with the chronic endometritis negative group.
      ,
      P < 0.001 embryo transfer/FET (%) compared with the chronic endometritis re-examination group.
      34.240<0.001
      Number and type of embryos transferred27.308<0.001
      Proportion of single cleavage-stage embryo transfer, % (n)5.36 (147/2742)4.96 (46/927)5.99 (20/334)
      Proportion of double cleavage-stage embryo transfer, % (n)49.12 (1347/2742)46.17 (428/927)60.78 (203/334)
      P < 0.001 the proportion of double cleavage stage embryo transfer (%) compared with the chronic endometritis-negative group.
      ,
      P < 0.001 the proportion of double cleavage-stage embryo transfer compared with the chronic endometritis re-examination group.
      Proportion of SBT, % (n)27.90 (765/2742)28.48 (264/927)17.96 (60/334)
      P < 0.001 the proportion of SBT (%) compared with the chronic endometritis negative group.
      ,
      P < 0.001 the proportion of SBT compared with the chronic endometritis re-examination group. DBT, double blastocyst transfer; proportion of DBT = cycles of DBT/total cycles of embryo transfer; proportion of double cleavage-stage embryo transfer = cycles of double cleavage-stage embryo transfer/total cycles of embryo transfer; embryo transfer/FET (n/n): cycle numbers of fresh embryos transferred/cycle numbers of frozen-thawed embryos transferred; FET, frozen embryo transfer; SBT, single blastocyst transfer; proportion of SBT = cycles of SBT/total cycles of embryo transfer; proportion of single cleavage-stage embryo transfer = cycles of single cleavage-stage embryo transfer/total cycles of embryo transfer.
      Proportion of DBT % (n)17.61 (483/2742)20.39 (189/927)15.27 (51/334)
      Endometrial thickness on the day of embryo transfer9.83 ± 2.189.91 ± 2.1610.00 ± 2.391.2170.296
      Embryo implantation rate, % (n)45.94 (2104/4580)46.27 (714/1543)46.85 (275/587)0.1990.905
      Clinical pregnancy rate % (n)58.83 (1613/2742)59.87 (555/927)59.30 (198/334)0.3180.853
      Early pregnancy loss rate, % (n)12.34 (199/1613)14.59 (81/555)12.63 (25/198)1.8870.389
      Late pregnancy loss rate, % (n)2.79 (45/1613)3.78 (21/555)3.54 (7/198)1.5110.470
      Ectopic pregnancy rate, % (n)1.80 (29/1613)3.06 (17/555)4.04 (8/198)5.9580.051
      Live birth rate49.09 (1346/2742)47.46 (440/927)47.60 (159/334)0.8720.647
      a P < 0.001 the embryo transfer/FET (%) compared with the chronic endometritis negative group.
      b P < 0.001 embryo transfer/FET (%) compared with the chronic endometritis re-examination group.
      c P < 0.001 the proportion of double cleavage stage embryo transfer (%) compared with the chronic endometritis-negative group.
      d P < 0.001 the proportion of double cleavage-stage embryo transfer compared with the chronic endometritis re-examination group.
      e P < 0.001 the proportion of SBT (%) compared with the chronic endometritis negative group.
      f P < 0.001 the proportion of SBT compared with the chronic endometritis re-examination group.DBT, double blastocyst transfer; proportion of DBT = cycles of DBT/total cycles of embryo transfer; proportion of double cleavage-stage embryo transfer = cycles of double cleavage-stage embryo transfer/total cycles of embryo transfer; embryo transfer/FET (n/n): cycle numbers of fresh embryos transferred/cycle numbers of frozen-thawed embryos transferred; FET, frozen embryo transfer; SBT, single blastocyst transfer; proportion of SBT = cycles of SBT/total cycles of embryo transfer; proportion of single cleavage-stage embryo transfer = cycles of single cleavage-stage embryo transfer/total cycles of embryo transfer.

      Comparison of maternal characteristics and pregnancy outcomes of chronic endometritis-positive groups after the first cycle of doxycycline treatment

      The chronic endometritis-positive groups treated with doxycycline and re-examined included 927 cycles. They were further divided into an improved chronic endometritis group (ICE) (n = 733) and a not-improved chronic endometritis group (NICE) (n = 194). No significant differences were observed between the two groups in age, years of infertility, body mass index, AMH level, basic FSH level, average number of high-quality embryos, percentage of embryo transfer and FETs, and number and type of embryos transferred. No significant differences were found in endometrial thickness on the day of embryo transfer, early pregnancy loss rate, late pregnancy loss rate, and ectopic pregnancy rate, whereas the embryo implantation rate, clinical pregnancy rate, and live birth rate in the NICE group was significantly lower than that in the ICE group (P = 0.008 and P = 0.001, respectively) (Table 3).
      TABLE 3COMPARISON OF MATERNAL CHARACTERISTICS AND PREGNANCY OUTCOMES AMONG THE CHRONIC ENDOMETRITIS POSITIVE GROUPS AFTER THE FIRST CYCLE OF DOXYCYCLINE ADMINISTRATION
      CharacteristicsImproved chronic endometritis groupNot-improved chronic endometritis groupF/χ2 valueP-value
      Cycles, n733194
      Age, years32.51 ± 4.4132.66 ± 4.874.8100.697
      Years of infertility3.68 ± 3.103.52 ± 2.652.8980.488
      BMI, kg/m221.95 ± 2.8521.86 ± 2.860.1110.703
      AMH, ng/ml3.54 (1.74–5.94)3.62 (1.88–5.76)0.664
      Basic FSH, IU/l6.71 (5.77–8.03)6.56 (5.73–7.63)0.195
      Average number of high-quality embryos6.79 ± 4.657.05 ± 4.450.0310.488
      Embryo transfer/FET, % (n)59.69 (274/459)42.65 (58/136)3.7370.053
      Number and type of embryos transferred4.3600.225
      Proportion of single cleavage-stage embryo transfer, % (n)27.69 (203/733)31.44 (61/194)
      Proportion of double cleavage-stage embryo transfer (%)4.64 (34/733)6.19 (12/194)
      Proportion of SBT, % (n)47.89 (351/733)39.69 (77/194)
      Proportion of DBT, % (n)19.78 (145/733)22.68 (44/194)
      Endometrial thickness on the day of embryo transfer9.98 ± 2.209.67 ± 2.001.5800.076
      Embryo implantation rate, % (n)47.64 (585/1228)40.95 (129/315)4.5080.0334
      Clinical pregnancy rate, % (n)62.07 (455/733)51.55 (100/194)7.7060.008
      Early pregnancy loss rate, % (n)8.73 (64/733)8.76 (17/194)0.0000.989
      Late pregnancy loss rate, % (n)1.91 (14/733)3.61 (7/194)1.3050.253
      Ectopic pregnancy rate (%)1.91 (14/733)1.55 (3/194)0.0010.972
      Live birth rate, % (n)50.20 (368/733)37.11 (72/194)10.5430.001
      Data presented as mean ± SD, % (n) and median (interquartile range).
      AMH, anti-Müllerian hormone; BMI, body mass index; FET, frozen embryo transfer; DBT, double blastocyst transfer; SBT, single blastocyst transfer.

      Logistic regression analysis of the live birth rate of infertile women with chronic endometritis treated with doxycycline

      Multivariate logistic regression analysis was conducted on the live birth rate of infertile women with chronic endometritis treated with doxycycline. As shown in Table 4, after adjusting for confounding factors, the differences in maternal age (adjusted OR 0.923, 95% CI 0.910 to 0.936), average number of high-quality embryos (adjusted OR 1.030, 95% CI 1.013 to 1.048), and endometrial thickness on the day of embryo transfer (adjusted OR 1.082, 95% CI 1.049 to1.116) were significant (P < 0.001). The live birth rate after embryo transfer at the single cleavage stage significantly decreased compared with that after SBT (adjusted OR 0.442, 95% CI 0.305 to 0.641), and the live birth rate after double blastocyst transfer (DBT) significantly increased compared with that after SBT (adjusted OR 1.676, 95% CI 1.376 to 2.041). No significant difference was found between the type of embryo transfer, i.e. SBT and double cleavage-stage embryo transfer (adjusted OR 1.095, 95% CI 0.920 to 1.303), and the cycle of embryo transfer or FET (adjusted OR 0.998, 95% CI 0.848 to 1.175). Compared with that in the chronic endometritis-negative group, the live birth rate in the chronic endometritis re-examination group (adjusted OR 0.895, 95% CI 0.767 to 1.045), and chronic endometritis non-re-examination group were not significantly different (adjusted OR 0.968, 95% CI 0.763 to1.230).
      TABLE 4LOGISTIC REGRESSION ANALYSIS OF THE LIVE BIRTH RATES OF INFERTILE WOMEN WITH CHRONIC ENDOMETRITIS TREATED WITH DOXYCYCLINE
      CharacteristicsUnadjusted odds ratio (95% CI)P-valueAdjusted odds ratio (95% CI)P-value
      Age, years0.916 (0.904 to 0.928)<0.0010.923 (0.910 to 0.936)<0.001
      Average number of high-quality embryos1.062 (1.048 to 1.077)<0.0011.030 (1.013 to 1.048)0.001
      Endometrial thickness on the day of embryo transfer1.702 (1.043 to1.101)<0.0011.082 (1.049 to 1.116)<0.001
      Embryo transfer or FET1.061 (0.938 to1.199)0.3460.998 (0.848 to 1.175)0.982
      Type of embryo transfer
      SBT1.001.00
      Single cleavage-stage0.334 (0.241 to 0.462)<0.0010.442 (0.305 to 0.641)<0.001
      Double cleavage-stage0.947 (0.825 to 1.807)0.4411.095 (0.920 to 1.303)0.308
      DBT1.602 (1.338 to 1.918)<0.0011.676 (1.376 to 2.041)<0.001
      Chronic endometritis-negative group1.001.00
      Chronic endometritis re-examination group0.937 (0.807 to 1.088)0.3930.895 (0.767 to 1.045)0.159
      Chronic endometritis non-re-examination group0.942 (0.751 to 1.183)0.6090.968 (0.763 to 1.230)0.791
      DBT, double blastocyst transfer; FET, frozen-thawed embryo transfer; SBT; single blastocyst transfer.
      Therefore, a higher maternal age and high proportion of embryos transferred in the single cleavage stage were considered risk factors for live birth rates in patients with chronic endometritis treated with doxycycline. In contrast, a high average number of high-quality embryos and high proportion of DBT were protective factors for live birth. Chronic endometritis endometrial non-re-examination, however, was not associated with live birth rates.

      Discussion

      This retrospective cohort study showed that endometrial re-examination of infertile women with chronic endometritis treated with doxycycline had no negative effect on pregnancy outcomes. The first cycle of doxycycline treatment could improve pregnancy outcomes in women with five or fewer CD138+ cells/HPF. To minimize the effect of confounding factors, we included patients who had two or more embryos available and adjusted variables, such as maternal age, cycle numbers of embryo transfer and FET, average number of high-quality embryos, and number and types of embryos transferred. Our results showed that the proportion of double cleavage-stage embryo transfer was significantly higher, whereas the proportion of SBT was significantly lower in the chronic endometritis non-re-examination group than in the other two groups. This can account for the higher proportion of fresh embryo transfer in the chronic endometritis non-re-examination group. The transfer of cleavage-stage embryos remains a priority in Chinese IVF centres, and the proportion of double cleavage-stage embryo transfer in the fresh embryo transfer cycle is higher than SBT. Studies (
      • Long X.
      • Wang Y.
      • Wu F.
      • Li R.
      • Chen L.
      • Qian W.
      • Qiao J.
      Pregnancy outcomes of single/double blastocysts and cleavage embryo transfers: a retrospective cohort study of 24,422 frozen-thawed cycles.
      ;
      • Zhu Q.
      • Lin J.
      • Gao H.
      • Wang N.
      • Wang B.
      • Wang Y.
      The association between embryo quality, number of transferred embryos and live birth rate after vitrified cleavage-stage embryos and blastocyst transfer.
      ) have shown that double cleavage-stage embryo transfer and SBT have similar pregnancy outcomes. This may explain why the pregnancy outcome in the chronic endometritis non-re-examination group was similar to that in the chronic endometritis-negative group and chronic endometritis-improved group. Live birth rates were not affected in women in the chronic endometritis re-examination group and chronic endometritis non-re-examination group.
      An increasing number of studies have revealed that chronic endometritis affected embryo implantation and reduced the pregnancy rate (
      • Kitaya K.
      • Takeuchi T.
      • Mizuta S.
      • Matsubayashi H.
      • Ishikawa T.
      Endometritis: new time, new concepts.
      ;
      • Kimura F.
      • Takebayashi A.
      • Ishida M.
      • Nakamura A.
      • Kitazawa J.
      • Morimune A.
      • Hirata K.
      • Takahashi A.
      • Tsuji S.
      • Takashima A.
      • Amano T.
      Review: Chronic endometritis and its effect on reproduction.
      ;
      • Buzzaccarini G.
      • Vitagliano A.
      • Andrisani A.
      • Santarsiero C.M.
      • Cicinelli R.
      • Nardelli C.
      • Ambrosini G.
      • Cicinelli E.
      Chronic endometritis and altered embryo implantation: a unified pathophysiological theory from a literature systematic review.
      ). Chronic endometritis is attributed to microbial infection of the uterine cavity. Pathogenic bacteria cannot be detected in more than one-half of the patients diagnosed with chronic endometritis combined with other infertility factors by bacterial culture methods and polymerase chain reaction (
      • Moreno I.
      • Simon C.
      Relevance of assessing the uterine microbiota in infertility.
      ). Currently, anti-infection treatment is considered a crucial method for the clinical treatment of chronic endometritis. Doxycycline is widely used because of its broad antibacterial spectrum and cost-effectiveness. Antibiotic treatment was reported as an effective way to cure chronic endometritis and improve reproductive outcomes. The prevalence of infertility in patients who received IVF/ICSI treatment in our hospital with five or more CD138+ cells/HPF in endometrial tissues was 31.50% (1261/4003), similar to the incidence of chronic endometritis in patients with infertility reported by
      • Cicinelli E.
      • Resta L.
      • Nicoletti R.
      • Zappimbulso V.
      • Tartagni M.
      • Saliani N.
      Endometrial micropolyps at fluid hysteroscopy suggest the existence of chronic endometritis.
      and
      • Kitaya K.
      • Yasuo T.
      Aberrant expression of selectin E, CXCL1, and CXCL13 in chronic endometritis.
      , which was 30.00% (45/150) and 28.95% (22/76), respectively. Our data showed that the cure rate of chronic endometritis was 79.07% (733/927) after the first cycle of oral antibiotic treatment and endometrial biopsy. Previous studies showed that cure rate of chronic endometritis ranged from 28.10% to 92.80% after one course of antibiotic treatment (
      • Johnston-MacAnanny E.B.
      • Hartnett J.
      • Engmann L.L.
      • Nulsen J.C.
      • Sanders M.M.
      • Benadiva C.A.
      Chronic endometritis is a frequent finding in women with recurrent implantation failure after in vitro fertilization.
      ;
      • Cicinelli E.
      • Matteo M.
      • Tinelli R.
      • Lepera A.
      • Alfonso R.
      • Indraccolo U.
      • Marrocchella S.
      • Greco P.
      • Resta L.
      Prevalence of chronic endometritis in repeated unexplained implantation failure and the IVF success rate after antibiotic therapy.
      ;
      • Kitaya K.
      • Matsubayashi H.
      • Takaya Y.
      • Nishiyama R.
      • Yamaguchi K.
      • Takeuchi T.
      • Ishikawa T.
      Live birth rate following oral antibiotic treatment for chronic endometritis in infertile women with repeated implantation failure.
      ;
      • Xiong Y.
      • Chen Q.
      • Chen C.
      • Tan J.
      • Wang Z.
      • Gu F.
      • Xu Y.
      Impact of oral antibiotic treatment for chronic endometritis on pregnancy outcomes in the following frozen–thawed embryo transfer cycles of infertile women: a cohort study of 640 embryo transfer cycles.
      ).
      A recent prospective randomized control trial (
      • Song D.
      • He Y.
      • Wang Y.
      • Liu Z.
      • Xia E.
      • Huang X.
      • Xiao Y.
      • Li T.C.
      Impact of antibiotic therapy on the rate of negative test results for chronic endometritis: a prospective randomized control trial.
      ) reported that the chronic endometritis cure rate after a 2-week course of antibiotics was 89.83% (53/59) compared with a spontaneous cure rate of 12.73% (7/55). Antibiotics can significantly reduce the detection rate of endometrial pathogenic microorganisms and increase the live birth rate (
      • Kitaya K.
      • Matsubayashi H.
      • Takaya Y.
      • Nishiyama R.
      • Yamaguchi K.
      • Takeuchi T.
      • Ishikawa T.
      Live birth rate following oral antibiotic treatment for chronic endometritis in infertile women with repeated implantation failure.
      ;
      • Huang W.
      • Liu B.
      • He Y.
      • Xie Y.
      • Liang T.
      • Bi Y.
      • Yuan L.
      • Qin A.
      • Wang Y.
      • Yang Y.
      Variation of diagnostic criteria in women with chronic endometritis and its effect on reproductive outcomes: A systematic review and meta–analysis.
      ). A recent retrospective study also showed that chronic endometritis was an independent risk factor for miscarriage, term delivery and live birth in patients with infertility (
      • Morimune A.
      • Kimura F.
      • Nakamura A.
      • Kitazawa J.
      • Takashima A.
      • Amano T.
      • Kaku S.
      • Moritani S.
      • Kushima R.
      • Murakami T.
      The effects of chronic endometritis on the pregnancy outcomes.
      ). Chronic endometritis induces a shift in the implantation window, affects the synchronization of the embryo and the inner membrane and influences implantation and pregnancy rates (
      • Kuroda K.
      • Horikawa T.
      • Moriyama A.
      • Nakao K.
      • Juen H.
      • Takamizawa S.
      • Ojiro Y.
      • Nakagawa K.
      • Sugiyama R.
      Impact of chronic endometritis on endometrial receptivity analysis results and pregnancy outcomes.
      ). Plasma cells may be present in the decidual tissues of patients diagnosed with chronic endometritis. The inflammation of the decidua is related to chronic endometritis, which leads to an increase in the miscarriage rate (
      • Kaku S.
      • Kubo T.
      • Kimura F.
      • Nakamura A.
      • Kitazawa J.
      • Morimune A.
      • Takahashi A.
      • Takebayashi A.
      • Takashima A.
      • Kushima R.
      • Murakami T.
      Relationship of chronic endometritis with chronic deciduitis in cases of miscarriage.
      ). A randomized controlled study (
      • Song D.
      • He Y.
      • Wang Y.
      • Liu Z.
      • Xia E.
      • Huang X.
      • Xiao Y.
      • Li T.C.
      Impact of antibiotic therapy on the rate of negative test results for chronic endometritis: a prospective randomized control trial.
      ) revealed that the continuous pregnancy and miscarriage rates between the chronic endometritis treatment group (27.12%, 3.39%) and chronic endometritis untreated control group (16.36%, 9.09%) did not show significant differences.
      A recent meta-analysis suggested that the effect of endometrial biopsy and injury on live birth and clinical pregnancy among women undergoing IVF is unclear (
      • Lensen S.F.
      • Armstrong S.
      • Gibreel A.
      • Nastri C.O.
      • Raine-Fenning N.
      • Martins W.P.
      Endometrial injury in women undergoing in vitro fertilisation (IVF).
      ). Notably, our study indicated that the clinical pregnancy and live birth rates of participants in the chronic endometritis non-re-examination group were similar to those in the chronic endometritis-negative and improved-chronic endometritis groups, which suggests that antibiotic treatment is highly effective. Most participants in the chronic endometritis non-re-examination group had direct fresh embryo transfer cycles and a higher proportion of double cleavage-stage embryo transfer. After antibiotic treatment, these patients could be subjected to IVF without endometrial biopsy and good pregnancy outcomes could be obtained. Therefore, our findings suggest that it is possible to reduce the pain and cost associated with endometrial biopsy without affecting live birth and clinical pregnancy among women undergoing IVF. This is the strength of this study.
      The present study, however, is limited by the retrospective and single-centre design, and thus our conclusion needs to be verified in further studies. More randomized controlled trials are needed to confirm the possible role of the microbiome and inflammation in patients with persistent chronic endometritis. The onset of chronic endometritis may be affected by other clinical and pathological factors, such as the effect of different endometrial sampling intervals on the diagnostic criteria of chronic endometritis and the outcome of assisted pregnancy. Therefore, further studies are necessary to clarify the relationship between plasma cell infiltration and chronic endometritis during different menstrual periods.
      In conclusion, endometrial re-examination of infertile women with chronic endometritis treated with doxycycline had no effect on pregnancy outcomes. The first cycle of doxycycline treatment could effectively improve the reproductive outcomes of women with five or more CD138+ cells/HPF. Notably, some infertile patients with chronic endometritis can achieve the benefit of improved pregnancy outcomes after receiving antibiotic treatment without endometrial re-examination.

      Acknowledgements

      The authors are grateful to Linjing Long for his support during the study.

      Funding

      The study was supported by a Guangdong Province Weiji Medical Develop's Fund (project number: K-202104-2). The sponsor had no role in study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the article for publication.

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      Biography

      Wenjuan Liu completed her master's degree in 2018 and has been practising reproductive medicine for 4 years. She specializes in female reproductive endocrinology and the treatment of infertility with chronic endometritis. She has published four papers in international journals and has participated in numerous domestic projects.
      Key message
      This large-sample retrospective cohort study suggests that endometrial re-examination of infertile women with chronic endometritis treated with doxycycline has no effect on pregnancy outcomes. The first cycle of doxycycline treatment could effectively improve the reproductive outcomes of women with five or more CD138+ cells/high-power field.