Characterization of uterine NK cells in women with infertility or recurrent pregnancy loss and associated endometriosis.

Method of study Mid-secretory phase endometrial biopsies were obtained and the endometrial expression of CD56, CD16 or NKp46 was identified by immunohistochemistry and quantified (ImageJ Software).

Materials and Methods Subjects

Sixty-one women were enrolled in the study. Twenty-one women had uRPL, 30 women had UI, and 10 women had no history of infertility, recurrent pregnancy loss, or endometriosis (controls). Among women with sub-fertility disorders, 13 women with uRPL and 23 with UI had concomitant endometriosis diagnosed by laparoscopic procedure performed in selected cases when clinical signs suggested the presence of the disease, either prior or post the endometrial biopsy obtained for this study. Sub-fertile subjects included women with UI and with 2 or more consecutive pregnancy losses (uRPL) who were evaluated in the Division of Reproductive Endocrinology at Greenville Hospital System in Greenville SC. Patients in the sub-fertility groups had regular, ovulatory cycles, at least one patent fallopian tube (without hydrosalpinges) and were 40 years of age or younger. Exclusion criteria included known uterine fibroids or septum, a history of pelvic inflammatory disease, PCOS or a partner with an abnormal semen analysis (by the World Health Organization (WHO) criteria). While a prior history of minimal or mild endometriosis was not an exclusion criterion, moderate or severe endometriosis was. Unexplained RPL were excluded if they had a known thrombophilia, genetic or immunologic abnormalities, or Müllerian defects. The fertile control group consisted of subjects who were patients in the Department of Obstetrics and Gynecology at University of North Carolina, Chapel Hill. All women who participated in the study signed an informed consent that was approved by the Institution Review Board (IRB) of Greenville Hospital System or University of North Carolina, Chapel Hill.

Tissue collection

All the participants underwent a standard endometrial biopsy by pipelle suction between days 21 to 24 of the menstrual cycle based on urinary LH surge detection (LH plus 7 to 10 days). Each patient was provided with a urinary LH detection kit (Sepal Inc., Boston, MA) and instructed to call the clinic with a positive LH surge. Tissue collected was submitted for paraffin embedding for histologic determination, dating and immunohistochemistry analysis. Women with maturational delay of endometrium (type I defect) were excluded from the study 23 , whereas a type 2 endometrial receptivity defect, based on the results of the αvβ3 integrin analysis, was found in 10 uRPL and 8 UI patients 24 .

Immunohistochemistry

The expression of CD56, CD16, or NKp46 in all endometrial samples was determined by immunohistochemistry. Endometrial samples were placed in 10% buffered formalin for 24 hours, dehydrated in graded ethanol, cleared in xylene and embedded in paraffin blocks. Briefly, the tissues in paraffin blocks were cut into 5 μm thick sections using the Leica RM 2135 microtome and transferred onto glass slides. Tissues were dewaxed in xylene and rehydrated through descending concentrated alcohols. Unmasking was performed in a 110V pressure cooker in a preheated 1:100 antigen unmasking solution (Vector Laboratories, Burlingame, CA) for 4 minutes. After washing, tissues were quenched in 3% hydrogen peroxide diluted in methanol for 10 minutes. Slides were then washed in 1% PBS and blocked in 10% NGS blocking buffer (Vector Laboratories, Burlingame, CA) for 1 hour at room temperature. Tissue sections were incubated with either monoclonal mouse anti-human CD56 (NCAM) antibody at 1:50 dilution (Novocastra Leica Biosystem, Buffalo Grove, IL, USA), monoclonal mouse anti-human CD16 antibody at a 1:20 dilution (AbD Serotec, Raleigh, NC, USA), or monoclonal goat anti-human NKp46/NCR1 antibody at 1:25 dilution (R&D Systems, Minneapolis, MN, USA) at +4°C overnight in a humidified chamber. Sections were washed extensively in 1% PBS and first incubated with the specific secondary antibody for 1 hour at room temperature followed by an incubation with a 1:1000 streptavidin HRP substrate for 30 minutes at room temperature. The positive cells were then visualized by incubation with DAB peroxidase solution (3.3′diaminobenzidene terahydrochloride; Vector Laboratories, Burlingame, CA). Slides were counterstained with haematoxylin (Biocare Medical), dehydrated in ascending concentrated alcohols, cleared in xylene and mounted in Permount (Fisher Scientific). Replacing CD56, CD16, NKp46 or LIF antibodies with an equivalent amount of control mouse IgG (Serotec) resulted in absence of immunoreactivity. Decidual tissue and liver tissues from baboons were used as positive controls at the same dilutions.

Analysis of images

Staining was assessed by using Olympus BH2 microscope (Olympus, Melville, NY, USA) and 10 high-resolution images of each specimen were captured at 400x magnification with a Nikon DS Fi1 digital camera/NIS Elements imaging software. The ratio between positive CD16, CD56 or NKp46 cells (brown stain) and total endometrial stromal cells (blue stain) was assessed using computer assisted image analysis with color deconvolution (Image J software, NIH) for all the images. The investigators were blinded with respect to the identification of the sections.

Statistical analysis

Data are expressed as average ± standard deviation of the mean (SD). Mann-whitney test was used to compare data from women with uRPL or UI with data from fertile control patients. Median values were calculated. Statistical significance was set at P