Pro-inflammatory Cytokines and Implantation Process in Women With Primary Idiopathic Infertility
Condition: Infertility, Female; Infertility Unexplained; Inflammatory Response
MeSH terms: Infertility; Infertility, Female
Study type: Observational

Summary: 
      Infertility is a common, worldwide problem. In about 20% of couples, the causative agent of
      infertility cannot be identified after routine diagnostic tests. One of the causes of
      idiopathic infertility may be implantation disorders.

      Implantation can take place at a strictly defined moment in the menstrual cycle, when the
      capacity of the blastocyst to implant is overlapped with readiness for its acceptance by the
      endometrium, the so-called endometrial receptivity.The time interval in which the endometrium
      exhibits this property is called the implantation window.

      The acquisition of receptivity by the endometrium is reflected in cellular and structural
      changes.The changes taking place at the cellular and molecular levels within the endometrium
      are compared to processes such as wound healing and degradation of the matrix during the
      neoplastic process.In considering the role of local inflammation in fertility, it is
      essential to distinguish between acute and chronic inflammation of moderate or low
      intensity.The profile of the molecules seen in a given inflammation depends on the severity,
      duration and mechanisms involved in the inflammation process, as well as the ability of the
      body's immune system to respond and adapt.IL-18 is a pro-inflammatory cytokine that mediates
      a type 1 cellular response. In the context of fertilization, IL-18 is a bivalent cytokine.
      Outside of the implantation window, IL-18 acts as an IFN-gamma inducer and is seen as a
      detrimental factor in the implantation process. During the implantation window, IL-18 becomes
      one of the main factors involved in the proper preparation of the spiral arteries. Histamine
      meets all the criteria of an inflammatory mediator. Histamine expression is also expressed in
      the endometrium, where it plays the role of a paracrine messenger during embryo
      decision-making and implantation. Adequate glucose uptake and metabolism are essential for
      the proper differentiation of the uterine endometrium towards a receptive state that allows
      the implantation of the embryo. The best described and most abundant glucose transporter in
      the endometrial stroma is GLUT1. However, there are no data on the role of GLUT4 in
      undetermined infertility.

      GLUT4 is one of the better studied transporters because of its major role in whole body
      glucose homeostasis and the pathogenesis of type II diabetes.

      Aims:1. Analysis of the level of interleukin 18 and histamine as molecules with a postulated
      role in the implantation process in the receptive endometrium in women with primary
      infertility of unknown etiology and comparing it to the group of women with naturally
      conceived offspring.

      2. Assessment of the correlation of the levels of interleukin 18 and histamine in the
      receptive endometrium and in the blood as an attempt to find a diagnostic useful marker of
      receptivity.

      3. Analysis of GLUT4 level in the receptive endometrium between two groups.

      Materials and Methods:

      Patients recruited from among women hospitalized at the CMUJ Gynecological Endocrinology
      Clinic for hormonal diagnostics.

        1. The patient's visit during the implantation window (appropriate time of the cycle
           determined on the basis of ultrasound ovulation monitoring)

        2. Endometrial aspiration biopsy, venous blood collection (5 ml). Preparation of material.

        3. Analysis of the collected material.
    

Detailed description: 
      Infertility affects approximately 8 to 12% of reproductive age couples worldwide, equivalent
      to more than 186 million people.

      In approximately 20% of couples, the causative agent of infertility cannot be identified
      after routine diagnostic tests. One of the causes of idiopathic infertility may be disorders
      of the implantation process.

      Implantation can take place at a specific point in the menstrual cycle, when the capacity of
      the blastocyst to implant is overlapped with readiness for its acceptance by the endometrium,
      the so-called endometrial receptivity.

      The time interval in which the endometrium exhibits this property is called the implantation
      window.

      The implantation window in humans is a period of 3 to 6 days, it falls on half of the
      secretory phase - that is, 6 to 10 days after the LH peak.

      The acquisition of receptivity by the endometrium is reflected in cellular and structural
      changes.

      The processes are hormonally controlled by estrogens and progesterone of ovarian origin. In
      addition, locally produced signaling molecules such as cytokines, growth factors, and lipid
      mediators via autocrine, paracrine and juxtacrine signaling are important.

      Genetically receptive and decisive are driven by the Hoxa10 and Hoxa11 homeobox genes, whose
      expression is increased by the action of steroid hormones. Changes at the cellular and
      molecular levels within the endometrium are compared to processes such as wound healing and
      degradation of the matrix during the process of cancer invasion.

      Among the cells of the immune system infiltrating the endometrium, mononuclear cells
      dominate: macrophages, lymphocytes and NK cells.

      In considering the role of local inflammation in fertility, it is essential to distinguish
      between acute and chronic inflammation of moderate or low intensity.

      In the acute phase of the inflammatory response, cells of the immune system migrate to the
      site of injury in a carefully planned sequence of events facilitated by soluble mediators
      such as cytokines, chemokines, and acute phase proteins. Depending on the severity of the
      injury, this acute phase may be sufficient to repair the damage and initiate healing
      processes.

      Prolonged inflammation, as a result of prolonged exposure to stimulation or an inappropriate
      response against one's own tissues, can lead to a chronic phase, in which tissue damage and
      fibrosis can occur.

      The profile of the molecules seen in a given inflammation depends on the severity, duration
      and mechanisms involved in the inflammation process, as well as the ability of the body's
      immune system to respond and adapt.

      Aim of the study was to investigate whether the levels of selected inflammatory molecules -
      interleukin18 and histamine, differ in the population of women with undetermined infertility
      compared to women with naturally conceived offspring.

      Materials and methods A prospective, cross-sectional study was conducted. The study design
      received a positive opinion of the Bioethics Committee of the Jagiellonian University No.
      1072.6120.78.2017 dated June 29, 2017 The funds were obtained from the grant No. K / DSC /
      00421 of the Ministry of Science and Higher Education. The study group consisted of 58 women
      diagnosed with primary infertility, and a control group consisted of 8 patients with
      naturally conceived offspring. Patients were recruited from among women hospitalized at the
      Gynecological Endocrinology and Gynecology Clinic, consulted at the Gynecological
      Endocrinology Clinic and diagnosed under the Comprehensive Reproductive Health Program in the
      period from November 2017 to March 2020.

      The inclusion criteria for the study group were met by women of reproductive age (18-40 years
      old) suffering from primary infertility (unsuccessfully trying to become pregnant for more
      than a year), menstruating regularly, not using hormone therapy.

      The exclusion criteria were: established male factor of infertility, tubal factor,
      endometriosis, inflammation in the pelvic area - currently and in the history, hormonal
      background of infertility, obesity, insulin resistance, status post at least one miscarriage
      and chronic autoimmune diseases.

      The inclusion criteria for the control group were met by patients of reproductive age (18-40
      years) with naturally conceived offspring up to three years of age, with regular
      menstruation. The exclusion criteria were: condition after surgery on the uterus, including
      curettage of the uterine cavity, endometriosis, pelvic inflammation - present and in the
      history, at least one miscarriage, obesity, insulin resistance, chronic autoimmune diseases,
      breastfeeding.

      Patients from both groups recruited for the study were examined during the implantation
      window. The appropriate timing of the cycle was determined by ultrasound ovulation
      monitoring. Patients reported around day 10 of the cycle to observe the growth of the
      dominant follicle. The examination was repeated after 3-4 days. If the dominant follicle
      growth was confirmed, the patient performed a urine LH test at home 16 days before the
      expected onset of menstruation. Patients reported 6-8 days after the LH peak or, in case of
      questionable test result, in the middle of the luteal phase determined by the reported length
      of the menstrual cycles - 8-4 days before the day of the expected period. During the visit,
      an ultrasound examination was performed to confirm past ovulation (observation of the corpus
      luteum in an identical ovary, endometrium at least 10 mm wide).

      The patients were informed about the necessity to give up intercourse in the cycle in which
      the study was conducted, which was confirmed in the study documentation. Patients who did not
      accept this requirement could not be admitted to the study. During the visit, after ovulation
      was confirmed, endometrial aspiration biopsy was performed using a plastic pipette with a
      diameter of approx. 3 mm (Pipelle Endometrial Suction Curette). If there were difficulties in
      penetrating the cervical canal with a pipette, the upper lip of the cervix was gripped with a
      clamp. In the case of severe pain in the patient or the inability to penetrate the cervical
      canal, collection of the material was discontinued. Immediately after the examination,
      patients were asked to rate the intensity of pain during the procedure numerically on the
      numerical rating scale (NRS), where 0 is no pain at all and 10 is the worst pain imaginable.
      There were no complications in any of the patients. The collected endometrial sample was
      preserved in a solution of 10% neutral, buffered formalin at room temperature, and then
      transferred to the CMUJ Clinical and Experimental Pathology Department for fixation in
      paraffin blocks.

      On the same day, a 5 ml venous blood sample on EDTA was collected from each patient. The
      blood was centrifuged for 15 minutes at 3000 rpm, and the serum was sent to the Department of
      Clinical Biochemistry of the Jagiellonian University Medical College for the determination of
      IL-18 and histamine levels.

      Immunohistochemistry Tissue material was fixed in formaldehyde for 24 hours. Then it was
      dehydrated in 70% ethanol solution and then embedded in paraffin. Tissues were sectioned 4 µm
      thick and mounted on silanized slides. The sections were divided into three equal sets.
      Paraffin preparations were dewaxed (3 xylene exchanges at room temperature; the first
      exchange - 10 minutes, the second and third exchange after 15 minutes) and rehydrated (3
      alcohol exchanges at room temperature, 5 minutes each). Then the preparations were incubated
      at room temperature for 10 min. in a 3% H2O2 solution to block endogenous peroxidase. Slides
      were rinsed in distilled water and incubated with TBS for 5 minutes. Then the slides were
      unmasked with citrate buffer pH 6.0 in a water bath at 97 ° C. After the preparations were
      unmasked, the Ultra Vision Protein Block preparation (Thermo Scientific) was applied for 5
      min. at room temperature in a humid chamber. Then the appropriate antibody was applied and
      incubated at room temperature in a humid chamber for a specified time (Table 1). The first
      set of sections was incubated with IL-18 reactive rabbit polyclonal antibodies (Recombinant
      IL18 (Tyr37 ~ Asp193), Cloud-Clone Corp., 0.33 mg / ml Cat. No. PAA064HU01) at a dilution of
      1:00 for 60 min. The second set was incubated with histamine reactive rabbit polyclonal
      antibodies (Recombinant human histamine (Met1 ~ Ile163), Cloud-Clone Corp., 0.35mg / ml,
      catalog no. PAA927Ge01) at a dilution of 1: 100 for 30 min. The third set was incubated with
      rabbit polyclonal antibodies (Recombinant human GLUT4 (Met1 ~ Ile163) Cloud-Clone Corp.,
      catalog no. PAC023HU01) at a dilution of 1:50 for 30 min. Excess antibody was washed with TBS
      buffer and Post antibody blocking for Bright Vision plus (BrightVision + Goat anti-mouse /
      rabbit HRP - ImmunoLogic kit) was applied. We incubate in a humid chamber at room temperature
      for 20 minutes.

      Excess reagent was washed off with TBS buffer and a poly-HRP-GAMs / Rb IgG preparation
      (BrightVision + Goat anti-mouse / rabbit HRP - ImmunoLogic kit) was applied. Incubated at
      room temperature in a humid chamber for 30 minutes. Excess reagent was washed off with TBS
      buffer and DAB Quanto (Thermo Scientific) was applied. Incubated at room temperature in a
      humid chamber for about 5 minutes. Then, the excess reagent was rinsed with distilled water
      and the slides were colored with hematoxylin (Thermo Scientific). The preparations were
      dehydrated by carrying out a series of three changes of alcohol and xylene in increasing
      concentrations. Closed with a coverslip (CYTOSEAL by ThermoScientific). Expression of each
      cytokine was assessed semi-quantitatively as the number of positive cells per square
      millimeter in the analyzed sample, calculated using an image analyzer.

      IL-18 and histamine in the blood Serum IL-18 measurement was performed by ELISA using human
      recombinant IL-18 (Human IL-18 ELISA Kit, Biorbyt, catalog no. Orb50153). Blood samples and
      reagents were warmed to 37 ° C and then diluted 1: 2 with the diluent using 50 µL of buffer
      with complete and uniform mixing. The solution was dispensed 0.1 ml per well. The microliter
      plate with the wells was covered with a lid and incubated at 37 ° C for 90 min. The plate was
      then dried, 0.1 ml of biotinylated anti-human IL-18 antibody solution was added to each well
      and incubated at 37 ° C for 60 min. The plate was then washed three times in 0.01 M TBS (TRIS
      buffered saline), each time leaving the wash buffer in the well for 1 min. The buffer was
      removed and the plate was dried, then 0.1 ml of prepared avidin-biotin-peroxidase (ABC)
      solution was added to each well and the plate was incubated at 37 ° C for 30 min. The plate
      was rinsed again, this time 5 times with 0.01 M TBS, and the washing buffer was left in the
      wells for 1-2 min each time. The buffer was removed and the plate was dried, then 90 µl of
      TMB dye was added to each well and the plate was incubated at 37 ° C for 10 min. 0.1 ml of
      TMB contrast dye solution was added to each well and the absorbance was immediately measured
      at 450 nm. Serum histamine levels were measured by ELISA using the Histamin ELISA
      Immundiagnostik reagent, catalog number K 8212. The washing buffer concentrate (WASHBUF B)
      was diluted 1:10 in pure water. Reagents were brought to room temperature. The plasma samples
      were mixed with the derivatization reagent. The derivatized sample was then incubated on a
      horizontal shaker together with a peroxidase labeled polyclonal histamine antibody on a
      histamine derivative coated ELISA plate for 1 hour at room temperature. Then the contents of
      the wells were washed 5 times with 250 µl of the washing buffer. After the last step,
      residual buffer was removed with tissue paper. Then 100 μl of substrate (SUB) was pipetted
      into each well, incubated for 15 minutes at room temperature, protected from light. 100 µl of
      the contrast dye solution was added to each well. The absorbance was measured immediately at
      450 nm. The results are expressed in picograms per milliliter.

      Statistical analysis. Statistical analysis was performed using the Statistica program
      (StatSoft 13.3). The Shapiro-Wilk normality test of the examined variables and the presented
      frequency charts showed that the distribution of the variables was not normal. The
      non-parametric Wald-Wolfowitz and U Mann-Whitney tests were used to compare the study group
      with the control group. The significance test of the correlation coefficient was based on the
      statistics for the Student's t-distribution.
    

Intervention name(s): Pipelle biopsy
Intervention type(s): Diagnostic Test
Intervention description(s): Ultrasonographically confirmed implantation window. Collection of material with use of Pipelle biopsy. Collection of 5 ml venous blood.
Other intervention name(s): Blood sampling
Observational model: Case-Control
Time perspective: Prospective
Sampling method: Non-Probability Sample
Study population: 
        The study group consisted of 58 women diagnosed with primary infertility, and a control
        group consisted of 8 patients with naturally conceived offspring. Patients were recruited
        from among women hospitalized at the Gynecological Endocrinology and Gynecology Clinic,
        consulted at the Gynecological Endocrinology Clinic and diagnosed under the Comprehensive
        Reproductive Health Program
      
Biospecimen description: 
      Endometrial tissue - aspiration biopsy Blood sample 5ml
    
Keywords: infertility; inflammation; endometrial receptivity