European Journal of Obstetrics & Gynecology and Reproductive Biology 181 (2014) 171–175
Contents lists available at ScienceDirect
European Journal of Obstetrics & Gynecology and Reproductive Biology journal homepage: www.elsevier.com/locate/ejogrb
The prognostic value of uNK cell count and histological dating in the mid-luteal phase of women with reproductive failure Beiyu Liu a,b, Najat Mariee a, Susan Laird c, John Smith d, Jie Li b, T.C. Li a,* a
Department of Reproductive Medicine, University of Sheffield, Sheffield Teaching Hospitals, Jessop Wing, Tree Root Walk, Sheffield S10 2SF, UK Reproductive Medicine Center, the First Affiliated Hospital of Sun Yat-sen University, Guangzhou, People’s Republic of China c Biomedical Research Centre, Sheffield Hallam University, Sheffield, UK d Department of Histopathology, Sheffield Teaching Hospitals, Sheffield, UK b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 15 October 2013 Received in revised form 16 June 2014 Accepted 20 July 2014
Objective: Histological dating has been used for decades to evaluate the histological maturation of the endometrium. Uterine natural killer cells are thought to play a significant role in pregnancy. While several studies have shown an increased number of uNK cells in women with recurrent reproductive failure, its prognostic value of pregnancy outcome remains uncertain. The aim of this study was to determine whether or not the prognostic value of uNK measurement on pregnancy outcome is improved when it is combined with histological dating of the same endometrial specimen. Study design: This is a retrospective study. Histological dating and uNK cell count was performed on endometrial biopsies taken from women with either recurrent miscarriage (RM, n = 94) or recurrent implantation failure (RIF, n = 72). Women who conceived within a year of the biopsy (n = 83) were included in a further analysis to examine the prognostic value of uNK cell count and histological dating on the outcome of a subsequent pregnancy. Results: There was a significant (p = 0.01) association between uNK cells and histological dating; retarded endometrium was less likely to have a high uNK cell count (16/58, 28%) than normally developed endometrium (52/108, 48%). Whilst uNK cell count on its own did not have a significant correlation to the pregnancy outcome, a retarded endometrium is significantly (p = 0.01) associated with a higher miscarriage rate (68%, 13/19) than normally developed endometrium (35%, 23/64) in women with reproductive failure. Regardless of the result of uNK cell count (normal or abnormal), combining the results of histological dating appeared to have significantly improved the prognostic value. Conclusions: We found that the prognostic value of uNK cell count is significantly increased when the result is combined with histological dating, primarily because histological dating is significantly correlated with pregnancy outcome. ß 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: Histological dating Recurrent implantation failure Recurrent miscarriage Natural killer cells Endometrium
Introduction The endometrium plays an important role in embryo implantation. Abnormal endometrial function leads to a spectrum of reproductive failure including infertility, miscarriage and late pregnancy complication. Currently, 50% of cases of recurrent reproductive failure (recurrent miscarriages (RM) as well as recurrent implantation failure are of unknown etiology, and an altered endometrial environment is thought to be associated with this idiopathic condition [1,2].
* Corresponding author. E-mail address: [email protected] (T.C. Li). http://dx.doi.org/10.1016/j.ejogrb.2014.07.010 0301-2115/ß 2014 Elsevier Ireland Ltd. All rights reserved.
Uterine natural killer cells are thought to play a significant role in the establishment and maintenance of early pregnancy. The phenotype of uterine NK cells differs from the peripheral blood NK cells. Ninety percent of peripheral blood NK cells are CD56+ CD16+ cells and are cytotoxic in nature. In contrast, 90% of the uNK population is of the CD56+ CD16 phenotype, which has little cytotoxic activity, but are a rich source of many different cytokines and growth factors [3]. Although their exact function in implantation remains unknown, several studies have demonstrated significant roles for uNK cells in immunotolerance, trophoblast invasion and angiogenesis [4–6]. The number of CD56+ cells in the endometrium varies through the menstrual cycle. There are few present in the proliferative phase, with numbers increasing in the mid- and late secretory phase of the menstrual cycle and further increasing in early pregnancy [7].
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Several studies have reported that uNK cell numbers in the endometrium during the peri-implantation period are elevated in women with idiopathic recurrent miscarriage [8,9] and recurrent implantation failure [10] as compared to control subjects. Nevertheless, the clinical value of uNK cell count in women with reproductive failure is uncertain, given that an earlier study [9] found that uNK cell measurement did not usefully predict pregnancy outcome in women with RM. During the luteal phase, the morphological changes observed in the endometrium occur in a highly predictable pattern [11,12]. Histological dating of the endometrium has been used for decades to evaluate the histological maturation of the endometrium and to diagnose luteal phase defect, which is defined as the retardation of endometrium development by more than 2 days from that expected from the chronological date based on the LH surge [13]. Theoretically, an inadequately developed endometrium can contribute to implantation failure which may manifest as infertility or pregnancy loss. However, the value of routine histological evaluation of the luteal phase endometrium in women with infertility is controversial [14,15]. Despite this, the prognostic value of histological evaluation of the endometrium on the outcome of a subsequent pregnancy (live birth or miscarriage) has not been formally examined. In this study, we conducted a retrospective analysis on a group of women with reproductive failure (either RIF or RM), who had endometrial biopsies taken for uNK cell measurement and became pregnant following biopsy, to examine the hypothesis that the prognostic value of uNK measurement on pregnancy outcome is improved when it is combined with histological dating of the same specimen.
microwave oven until boiling. Slides were added to the buffer and left covered at high heat for 3 min. Slides were further incubated for 12 min in the heated buffer before being transferred to cool citrate buffer for 20 min. An ABC kit (Vector Laboratories, UK) was used according to the manufacturer’s instructions with some adaptations. Slides were washed in TBS and blocked in TBS plus normal horse serum containing 250 ml avidin/ml (Vector Laboratories) for 1 h at room temperature, and incubated overnight at 4 8C with a mouse monoclonal primary anti-CD56 antibody (NCL-CD56504; Novacastra Laboratories Ltd, UK) diluted 1:50 in antibody buffer (TBS plus normal horse serum) containing 250 ml/ml biotin. Slides were washed in TBS throughout, and after application of secondary antibody and Vectorstain, binding was visualized by incubation with peroxidase substrate DAB (3.30 diaminobenzidene terahydrochloride; Vector Laboratories). Slides were washed in distilled water and counterstained with 20% hematoxylin for 30 min, differentiated, dehydrated through the alcohols, cleared in xylene and mounted in Vectormount (Vector Laboratories).
Materials and Methods
Histological dating
Subjects
Histological dating was carried out by either of the two investigators (J.S. and T.C.L) who are experienced in the use of the histological dating criteria of Noyes et al. [11]. They were not aware of the results of NK cells count at the time of histological dating. The results were considered abnormal if histological dating was >2 days behind chronological dating [13].
This study was primarily based on archived endometrial specimens. Most of them were collected as part of previous studies on the role of endometrium in reproductive failure [9,10,16,17]. Women included in the study were recruited from the recurrent miscarriage clinic and assisted conception unit of the Jessop Wing, Sheffield Teaching Hospitals, Sheffield, UK. They all had endometrial biopsies taken in the mid-luteal phase for uNK cell count. The biopsies were obtained from a total of 94 women with recurrent miscarriage (three or more consecutive miscarriages in the first trimester of pregnancy) and 72 women with unexplained recurrent implantation failure (defined as failure to achieve a clinical pregnancy after three or more IVF/embryo replacement cycles in which 4 or more good quality embryos had been replaced). Women were advised not to have unprotected sexual intercourse during the biopsy cycle to avoid having a biopsy in the conception cycle. All women had daily urine LH measurements from day nine onwards to identify the LH surge. The endometrial biopsies were collected using a Pipelle sampler (Prodimed, France) on days 7–9 after the LH surge, in the outpatients department. Immunohistochemistry As soon as the biopsy specimen was obtained, it was fixed overnight in formalin and automatically wax embedded for immunohistochemistry staining. Five micrometer sections of endometrial tissue were cut and dewaxed in xylene, rehydrated through the alcohols to tris-buffered saline (TBS) (pH 7.6) and quenched in 3% hydrogen peroxide in methanol for 20 min. After washing, unmasking was performed in an 800 W microwave oven in 10 mmol/l citrate buffer (pH 6.0). Buffer was heated in the
Counting of uNK cells The counting of uNK cells was carried by N.M. The number of positively stained uNK cells and the number of negatively stained stromal cells were counted in 10 random non-overlapping microscope fields at a magnification of 400. Cell number was expressed as a percent of total stromal cells. A uNK cell count of more than 13.9% was considered abnormally high [9]. All biopsies contained sufficient tissue to enable at least 3800 stromal cells to be examined, which we have shown previously to be necessary for accurate assessment of uNK cell number [18].
Pregnancy outcome The medical records of the patients were reviewed to determine if conception occurred following the biopsy. When conception did occur, the outcome was categorized into four groups: (1) Loss prior to detection of fetal heart beats, (2) fetal loss in the first trimester after fetal heart beats had been detected, (3) mid-trimester loss (loss between 13 and 24 weeks gestation) and (4) third-trimester loss (loss after 24 weeks). Women who conceived within a year of the biopsy were included in a further analysis to examine the prognostic value of uNK cell count and histological dating on the outcome of the subsequent pregnancy. In women who conceived more than once after the biopsy, only the first pregnancy after the biopsy was included. Four women who conceived more than 12 months after the endometrial biopsy was obtained were excluded. Two women who conceived whilst taking prednisolone for high NK cell count were also excluded. Statistical methods Data were analyzed using the Statistical Package for Social Science (SPSS) version 20. Pearson Chi-square was used to analyze the correlation of uNK cell measurement and histological dating, as well as the prognostic value of uNK cell measurement, histological dating and combined measurement. Differences were considered to be statistically significant when the p value was 26 w) Live birth
66 17 4.7 3.4
15 18 2 1 47
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Table 4 The prognostic value of uNK cell and histological dating on the outcome of pregnancy.
uNK cella Normal High Histological datingb Normal Abnormal
Miscarriage
Live birth
23 (44%) 13 (41%)
29 (56%) 18 (58%)
23 (36%) 13 (68%)
41 (64%) 6 (32%)
a
p = 0.84 (Pearson Chi-square). p = 0.01 (Pearson Chi-square). For histological dating, positive predictive value was 68.4%, negative predictive value was 64.1%. b
does histological dating result improve the prognostic value of uNK cell count on pregnancy outcome? Our finding suggests it does. Table 5 showed that the prognostic value of uNK cell count and histological dating combined is significant greater than uNK cell count. Our data showed that histological dating on its own is significantly correlated to pregnancy outcome. Women with normal endometrium development have a 65% (41/64) chance of a live birth, as compared to women with retarded endometrium who have a 32% (16/19) chance of a live birth. Theoretically, an inadequately developed endometrium during the implantation window can contribute to both infertility (lack of implantation) as well as recurrent pregnancy loss. The association between retarded endometrium and women with infertility and recurrent miscarriage has been reported [15,27]. The usefulness of routine histological dating as a screening test in the infertile population has been questioned in a previous study, as it was unable to discriminate between women who are fertile or infertile [14]. However, the prognostic value of histological dating on pregnancy outcome in women with reproductive failure has not been previously examined. The finding that histological dating of endometrium is of prognostic value in the outcome of a subsequent pregnancy in women with recurrent reproductive failure is therefore new. In this study, we have confined our observations to the relationship between uNK cell counts and histological dating and pregnancy outcome. We have not attempted to examine the relationship of the measurements to fecundity or the likelihood of conception. The main reason is that this group of women conceived with different treatment modalities, some spontaneously, some following assisted conception, and at different time intervals after the investigation. To examine the relationship between these two measurements and fecundity requires that they all receive the same treatment and the use of cumulative conception rate to adjust for the duration of follow up. Therefore, the finding in this study on the prognostic value of histological dating and/or uNK cell count on subsequent pregnancy outcome should not be extrapolated to the prediction of the likelihood of conception. Further
Table 5 The impact of histological dating results on the prognostic value of uNK cells count in women with reproductive failure. Miscarriage rate uNK normal All cases Normal histological datinga Abnormal histological datingb uNK high All cases Normal histological datingc Abnormal histological datingd a vs b, p = 0.07. c vs d, p = 0.02.
44% (23/52) 36% (13/36) 62% (10/16) 41% (13/31) 36% (10/28) 100% (3/3)
work is also necessary to determine whether or not histological dating has any prognostic value on pregnancy outcome in the general infertility population. In our study, we used two separate methods (uNK cell count and histological dating) in endometrium in peri-implantation period. We think our study highlights an important concept in the evaluation of endometrium in women with reproductive failure. The vast majority of, if not all, previous studies focused on a single marker to assess endometrial function. However, our study suggests that multiple evaluations is necessary to improve the clinical usefulness of the measurements, just as multiple markers have been successfully developed to screen for ovarian cancer, prostate cancer, Down’s syndrome, etc. Our study appears to be the first of its kind to use combined measurements to prognosticate pregnancy outcome. The predictive value of the combined measurement may be further improved if the karyotype result of the abortus in women with recurrent miscarriage is known. If the karyotype result of the abortus is normal, the likelihood of an underlying endometrial abnormality is increased compared with the situation when the karyotype of the abortus is abnormal, as the endometrium in such a case is more likely to be normal. However, we do not have in our series karyotype result of the preceding miscarriage in the majority of subjects to enable us to carry out the additional analysis. It is desirable in future studies to incorporate fetal karyotyping results to verify to what extent the additional information improves the predictive value. One possible criticism of our study is that we included two categories of women with reproductive failure, namely, women with RM and RIF. Although they do have some differences, they both appear to have an increased prevalence of increased uNK cell count [19] and a presumed uterine factor to account for their reproductive failure. Among the 166 women included in the study, 94 women had RM and 72 women had RIF. Among 94 women with RM, 69 (73%) conceived within a year of the investigation and fulfilled the inclusion criteria whereas among 72 women with RIF, only 14 (19%) conceived within a year of the investigation and fulfilled the inclusion criteria. The apparent discrepancy in the conception rate (hence the inclusion) relates to the different reproductive performance of the two groups: RM subjects do not have problem conceiving whereas women with RIF do. Furthermore, women with recurrent miscarriage conceived spontaneously whereas women with RIF had to undergo further IVF treatment and received luteal support during early pregnancy. Nevertheless, if we had confined the analysis of the prognostic value of uNK cell and histological dating only to women with RM (n = 64), similar results were obtained: there was no significant correlation (p = 0.9) between pregnancy outcome and uNK cell count but there was a significant (p = 0.05) correlation with histological dating. It should be noted that there are two populations of uNK in the human endometrium. The majority (90%) are CD56+ CD16, but there is a small population (10%) of CD56+ CD16+ cells which resemble peripheral blood NK cells, and some studies have suggested that it is this specific population of cells that is associated with reproductive failure [21,28]. In this study, we only looked at the whole population of uNK cells. In a previous study, we have shown that the numbers of CD56+ cells, but not CD16+ cells, were increased in women with recurrent implantation failure after IVF [10] and this was the reason for concentrating on this cell population in this study. Previous studies have suggested that specific populations of endometrial T cells, Treg cells, may be important in recurrent miscarriage [29]. However, these cells only make up a very small proportion of the endometrial leucocyte population and they are a less obvious marker for predicting pregnancy loss than the CD56+ cell population.
B. Liu et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 181 (2014) 171–175
To conclude, we found that the prognostic value of uNK cell is significantly increased when the result is combined with histological dating, primarily because histological dating is significantly correlated with pregnancy outcome. Condensation The prognostic value of uNK cell count on pregnancy outcome in women with reproductive failure is significantly increased when the result is combined with histological dating. References [1] Toth B, Wu¨rfel W, Germeyer A, Hirv K, Makrigiannakis A, Strowitzki T. Disorders of implantation—are there diagnostic and therapeutic options? J Reprod Immunol 2011;90:117–23. [2] Li TC, Makris M, Tomsu M, Tuckerman E, Laird S. Recurrent miscarriage: aetiology, management and prognosis. Hum Reprod Update 2002;8:463–81. [3] Vacca P, Moretta L, Moretta A, Mingari MC. Origin, phenotype and function of human natural killer cells in pregnancy. Trends Immunol 2011;32:517–23. [4] Quenby S, Nik H, Innes B, Lash G, Turner M, Drury J, et al. Uterine natural killer cells and angiogenesis in recurrent reproductive failure. Hum Reprod 2008;24:45–54. [5] Quenby S, Farquharson R. Uterine natural killer cells, implantation failure and recurrent miscarriage. Reprod Biomed Online 2006;13:24–8. [6] Wallace AE, Fraser R, Cartwright JE. Extravillous trophoblast and decidual natural killer cells: a remodelling partnership. Hum Reprod Update 2012;18: 458–71. [7] Lash GE, Robson SC, Bulmer JN. Review functional role of uterine natural killer (uNK) cells in human early pregnancy decidua. Placenta 2010;31:S87–92. [8] Tang AW, Alfirevic Z, Quenby S. Natural killer cells and pregnancy outcomes in women with recurrent miscarriage and infertility: a systematic review. Hum Reprod 2011;26:1971–80. [9] Tuckerman E, Laird SM, Prakash A, Li TC. Prognostic value of the measurement of uterine natural killer cells in the endometrium of women with recurrent miscarriage. Hum Reprod 2007;22:2208–13. [10] Tuckerman E, Mariee N, Prakash A, Li TC, Laird S. Uterine natural killer cells in peri-implantation endometrium from women with repeated implantation failure after IVF. J Reprod Immunol 2010;87:60–6. [11] Noyes RW, Hertig AT, Rock J. Dating the endometrial biopsy. Am J Obstet Gynecol 1975;122:262–3. [12] Li TC, Rogers AW, Dockery P, Lenton EA, Cooke ID. A new method of histologic dating of human endometrium in the luteal phase. Fertil Steril 1988;50:52–60. [13] Li TC, Cooke ID. Evaluation of the luteal phase. Hum Reprod 1991;6:484–99.
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[14] Coutifaris C, Myers ER, Guzick DS, et al. Histological dating of timed endometrial biopsy tissue is not related to fertility status. Fertil Steril 2004;82:1264–72. [15] Klentzeris LD, Li TC, Dockery P, Cooke ID. The endometrial biopsy as a predictive factor of pregnancy rate in women with unexplained infertility. Eur J Obstet Gynaecol Reprod Biol 1992;45:119–24. [16] Laird SM, Mariee N, Wei L, Li TC. Measurements of CD56+ cells in peripheral blood and endometrium by flow cytometry and immunohistochemical staining in situ. Hum Reprod 2011;26:1331–7. [17] Mariee N, Li TC, Laird SM. Expression of leukaemia inhibitory factor and interleukin 15 in endometrium of women with recurrent implantation failure after IVF; correlation with the number of endometrial natural killer cells. Hum Reprod 2012;27:1946–54. [18] Mariee N, Tuckerman E, Ali A, Li W, Laird S, Li TC. The observer and cycle-tocycle variability in the measurement of uterine natural killer cells by immunohistochemistry. J Reprod Immunol 2012;95:93–100. [19] Lash GE, Bulmer JN. Do uterine natural killer (uNK) cells contribute to female reproductive disorders. J Reprod Immunol 2011;88:156–64. [20] Lachapelle MH, Miron P, Hemmings R, Roy DC. Endometrial T, B, and NK cells in patients with recurrent spontaneous abortion—altered profile and pregnancy outcome. J Immunol 1996;156:4027–34. [21] Dosiou C, Giudice LC. Natural killer cells in pregnancy and recurrent pregnancy loss: endocrine and immunologic perspectives. Endocr Rev 2005;26: 44–62. [22] Croy BA, Zhang J, Tayade C, Colucci F, Yadi H, Yamada AT. Analysis of uterine natural killer cells in mice. Meth Mol Biol 2010;612:465–503. [23] Quenby S, Bates M, Doig T, et al. Pre-implantation endometrial leukocytes in women with recurrent miscarriage. Hum Reprod 1999;14:2386–91. [24] Clifford K, Flanagan AM, Regan L. Endometrial CD56+ natural killer cells in women with recurrent miscarriage: a histomorphometric study. Hum Reprod 1999;14:2727–30. [25] Ledee-Bataille N, Bonnet-Chea K, Hosny G, Dubanchet SY, Frydman R, Chaouat G. Role of the endometrial tripod interleukin-18, -15, and -12 in inadequate uterine receptivity in patients with a history of repeated in vitro fertilization– embryo transfer failure. Fertil Steril 2005;83:598–605. [26] Michimata T, Ogasawara MS, Tsuda H, et al. Distributions of endometrial NK cells, B, cells, T cells, and Th2/Tc2 cells fail to predict pregnancy outcome following recurrent abortion. Am J Reprod Immunol 2002;47: 196–202. [27] Serle E, Aplin JD, Li TC, et al. Endometrial differentiation in the peri-implantation phase of women with recurrent miscarriage: a morphological and immunohistochemical study. Fertil Steril 1994;62:989–96. [28] Laird SM, Tuckerman EM, Cork BA, Linjawi S, Blakemore AIF, Li TC. A review of immune cells and molecules in women with recurrent miscarriage. Hum Reprod Update 2003;9:163–74. [29] Kallikourdis M, Betz AG. Periodic accumulation of regulatory T cells in the uterus: preparation for the implantation of a semi-allogeneic fetus? PLoS One 2007;2:e382.
Contents lists available at ScienceDirect
European Journal of Obstetrics & Gynecology and Reproductive Biology journal homepage: www.elsevier.com/locate/ejogrb
The prognostic value of uNK cell count and histological dating in the mid-luteal phase of women with reproductive failure Beiyu Liu a,b, Najat Mariee a, Susan Laird c, John Smith d, Jie Li b, T.C. Li a,* a
Department of Reproductive Medicine, University of Sheffield, Sheffield Teaching Hospitals, Jessop Wing, Tree Root Walk, Sheffield S10 2SF, UK Reproductive Medicine Center, the First Affiliated Hospital of Sun Yat-sen University, Guangzhou, People’s Republic of China c Biomedical Research Centre, Sheffield Hallam University, Sheffield, UK d Department of Histopathology, Sheffield Teaching Hospitals, Sheffield, UK b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 15 October 2013 Received in revised form 16 June 2014 Accepted 20 July 2014
Objective: Histological dating has been used for decades to evaluate the histological maturation of the endometrium. Uterine natural killer cells are thought to play a significant role in pregnancy. While several studies have shown an increased number of uNK cells in women with recurrent reproductive failure, its prognostic value of pregnancy outcome remains uncertain. The aim of this study was to determine whether or not the prognostic value of uNK measurement on pregnancy outcome is improved when it is combined with histological dating of the same endometrial specimen. Study design: This is a retrospective study. Histological dating and uNK cell count was performed on endometrial biopsies taken from women with either recurrent miscarriage (RM, n = 94) or recurrent implantation failure (RIF, n = 72). Women who conceived within a year of the biopsy (n = 83) were included in a further analysis to examine the prognostic value of uNK cell count and histological dating on the outcome of a subsequent pregnancy. Results: There was a significant (p = 0.01) association between uNK cells and histological dating; retarded endometrium was less likely to have a high uNK cell count (16/58, 28%) than normally developed endometrium (52/108, 48%). Whilst uNK cell count on its own did not have a significant correlation to the pregnancy outcome, a retarded endometrium is significantly (p = 0.01) associated with a higher miscarriage rate (68%, 13/19) than normally developed endometrium (35%, 23/64) in women with reproductive failure. Regardless of the result of uNK cell count (normal or abnormal), combining the results of histological dating appeared to have significantly improved the prognostic value. Conclusions: We found that the prognostic value of uNK cell count is significantly increased when the result is combined with histological dating, primarily because histological dating is significantly correlated with pregnancy outcome. ß 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: Histological dating Recurrent implantation failure Recurrent miscarriage Natural killer cells Endometrium
Introduction The endometrium plays an important role in embryo implantation. Abnormal endometrial function leads to a spectrum of reproductive failure including infertility, miscarriage and late pregnancy complication. Currently, 50% of cases of recurrent reproductive failure (recurrent miscarriages (RM) as well as recurrent implantation failure are of unknown etiology, and an altered endometrial environment is thought to be associated with this idiopathic condition [1,2].
* Corresponding author. E-mail address: [email protected] (T.C. Li). http://dx.doi.org/10.1016/j.ejogrb.2014.07.010 0301-2115/ß 2014 Elsevier Ireland Ltd. All rights reserved.
Uterine natural killer cells are thought to play a significant role in the establishment and maintenance of early pregnancy. The phenotype of uterine NK cells differs from the peripheral blood NK cells. Ninety percent of peripheral blood NK cells are CD56+ CD16+ cells and are cytotoxic in nature. In contrast, 90% of the uNK population is of the CD56+ CD16 phenotype, which has little cytotoxic activity, but are a rich source of many different cytokines and growth factors [3]. Although their exact function in implantation remains unknown, several studies have demonstrated significant roles for uNK cells in immunotolerance, trophoblast invasion and angiogenesis [4–6]. The number of CD56+ cells in the endometrium varies through the menstrual cycle. There are few present in the proliferative phase, with numbers increasing in the mid- and late secretory phase of the menstrual cycle and further increasing in early pregnancy [7].
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B. Liu et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 181 (2014) 171–175
Several studies have reported that uNK cell numbers in the endometrium during the peri-implantation period are elevated in women with idiopathic recurrent miscarriage [8,9] and recurrent implantation failure [10] as compared to control subjects. Nevertheless, the clinical value of uNK cell count in women with reproductive failure is uncertain, given that an earlier study [9] found that uNK cell measurement did not usefully predict pregnancy outcome in women with RM. During the luteal phase, the morphological changes observed in the endometrium occur in a highly predictable pattern [11,12]. Histological dating of the endometrium has been used for decades to evaluate the histological maturation of the endometrium and to diagnose luteal phase defect, which is defined as the retardation of endometrium development by more than 2 days from that expected from the chronological date based on the LH surge [13]. Theoretically, an inadequately developed endometrium can contribute to implantation failure which may manifest as infertility or pregnancy loss. However, the value of routine histological evaluation of the luteal phase endometrium in women with infertility is controversial [14,15]. Despite this, the prognostic value of histological evaluation of the endometrium on the outcome of a subsequent pregnancy (live birth or miscarriage) has not been formally examined. In this study, we conducted a retrospective analysis on a group of women with reproductive failure (either RIF or RM), who had endometrial biopsies taken for uNK cell measurement and became pregnant following biopsy, to examine the hypothesis that the prognostic value of uNK measurement on pregnancy outcome is improved when it is combined with histological dating of the same specimen.
microwave oven until boiling. Slides were added to the buffer and left covered at high heat for 3 min. Slides were further incubated for 12 min in the heated buffer before being transferred to cool citrate buffer for 20 min. An ABC kit (Vector Laboratories, UK) was used according to the manufacturer’s instructions with some adaptations. Slides were washed in TBS and blocked in TBS plus normal horse serum containing 250 ml avidin/ml (Vector Laboratories) for 1 h at room temperature, and incubated overnight at 4 8C with a mouse monoclonal primary anti-CD56 antibody (NCL-CD56504; Novacastra Laboratories Ltd, UK) diluted 1:50 in antibody buffer (TBS plus normal horse serum) containing 250 ml/ml biotin. Slides were washed in TBS throughout, and after application of secondary antibody and Vectorstain, binding was visualized by incubation with peroxidase substrate DAB (3.30 diaminobenzidene terahydrochloride; Vector Laboratories). Slides were washed in distilled water and counterstained with 20% hematoxylin for 30 min, differentiated, dehydrated through the alcohols, cleared in xylene and mounted in Vectormount (Vector Laboratories).
Materials and Methods
Histological dating
Subjects
Histological dating was carried out by either of the two investigators (J.S. and T.C.L) who are experienced in the use of the histological dating criteria of Noyes et al. [11]. They were not aware of the results of NK cells count at the time of histological dating. The results were considered abnormal if histological dating was >2 days behind chronological dating [13].
This study was primarily based on archived endometrial specimens. Most of them were collected as part of previous studies on the role of endometrium in reproductive failure [9,10,16,17]. Women included in the study were recruited from the recurrent miscarriage clinic and assisted conception unit of the Jessop Wing, Sheffield Teaching Hospitals, Sheffield, UK. They all had endometrial biopsies taken in the mid-luteal phase for uNK cell count. The biopsies were obtained from a total of 94 women with recurrent miscarriage (three or more consecutive miscarriages in the first trimester of pregnancy) and 72 women with unexplained recurrent implantation failure (defined as failure to achieve a clinical pregnancy after three or more IVF/embryo replacement cycles in which 4 or more good quality embryos had been replaced). Women were advised not to have unprotected sexual intercourse during the biopsy cycle to avoid having a biopsy in the conception cycle. All women had daily urine LH measurements from day nine onwards to identify the LH surge. The endometrial biopsies were collected using a Pipelle sampler (Prodimed, France) on days 7–9 after the LH surge, in the outpatients department. Immunohistochemistry As soon as the biopsy specimen was obtained, it was fixed overnight in formalin and automatically wax embedded for immunohistochemistry staining. Five micrometer sections of endometrial tissue were cut and dewaxed in xylene, rehydrated through the alcohols to tris-buffered saline (TBS) (pH 7.6) and quenched in 3% hydrogen peroxide in methanol for 20 min. After washing, unmasking was performed in an 800 W microwave oven in 10 mmol/l citrate buffer (pH 6.0). Buffer was heated in the
Counting of uNK cells The counting of uNK cells was carried by N.M. The number of positively stained uNK cells and the number of negatively stained stromal cells were counted in 10 random non-overlapping microscope fields at a magnification of 400. Cell number was expressed as a percent of total stromal cells. A uNK cell count of more than 13.9% was considered abnormally high [9]. All biopsies contained sufficient tissue to enable at least 3800 stromal cells to be examined, which we have shown previously to be necessary for accurate assessment of uNK cell number [18].
Pregnancy outcome The medical records of the patients were reviewed to determine if conception occurred following the biopsy. When conception did occur, the outcome was categorized into four groups: (1) Loss prior to detection of fetal heart beats, (2) fetal loss in the first trimester after fetal heart beats had been detected, (3) mid-trimester loss (loss between 13 and 24 weeks gestation) and (4) third-trimester loss (loss after 24 weeks). Women who conceived within a year of the biopsy were included in a further analysis to examine the prognostic value of uNK cell count and histological dating on the outcome of the subsequent pregnancy. In women who conceived more than once after the biopsy, only the first pregnancy after the biopsy was included. Four women who conceived more than 12 months after the endometrial biopsy was obtained were excluded. Two women who conceived whilst taking prednisolone for high NK cell count were also excluded. Statistical methods Data were analyzed using the Statistical Package for Social Science (SPSS) version 20. Pearson Chi-square was used to analyze the correlation of uNK cell measurement and histological dating, as well as the prognostic value of uNK cell measurement, histological dating and combined measurement. Differences were considered to be statistically significant when the p value was 26 w) Live birth
66 17 4.7 3.4
15 18 2 1 47
174
B. Liu et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 181 (2014) 171–175
Table 4 The prognostic value of uNK cell and histological dating on the outcome of pregnancy.
uNK cella Normal High Histological datingb Normal Abnormal
Miscarriage
Live birth
23 (44%) 13 (41%)
29 (56%) 18 (58%)
23 (36%) 13 (68%)
41 (64%) 6 (32%)
a
p = 0.84 (Pearson Chi-square). p = 0.01 (Pearson Chi-square). For histological dating, positive predictive value was 68.4%, negative predictive value was 64.1%. b
does histological dating result improve the prognostic value of uNK cell count on pregnancy outcome? Our finding suggests it does. Table 5 showed that the prognostic value of uNK cell count and histological dating combined is significant greater than uNK cell count. Our data showed that histological dating on its own is significantly correlated to pregnancy outcome. Women with normal endometrium development have a 65% (41/64) chance of a live birth, as compared to women with retarded endometrium who have a 32% (16/19) chance of a live birth. Theoretically, an inadequately developed endometrium during the implantation window can contribute to both infertility (lack of implantation) as well as recurrent pregnancy loss. The association between retarded endometrium and women with infertility and recurrent miscarriage has been reported [15,27]. The usefulness of routine histological dating as a screening test in the infertile population has been questioned in a previous study, as it was unable to discriminate between women who are fertile or infertile [14]. However, the prognostic value of histological dating on pregnancy outcome in women with reproductive failure has not been previously examined. The finding that histological dating of endometrium is of prognostic value in the outcome of a subsequent pregnancy in women with recurrent reproductive failure is therefore new. In this study, we have confined our observations to the relationship between uNK cell counts and histological dating and pregnancy outcome. We have not attempted to examine the relationship of the measurements to fecundity or the likelihood of conception. The main reason is that this group of women conceived with different treatment modalities, some spontaneously, some following assisted conception, and at different time intervals after the investigation. To examine the relationship between these two measurements and fecundity requires that they all receive the same treatment and the use of cumulative conception rate to adjust for the duration of follow up. Therefore, the finding in this study on the prognostic value of histological dating and/or uNK cell count on subsequent pregnancy outcome should not be extrapolated to the prediction of the likelihood of conception. Further
Table 5 The impact of histological dating results on the prognostic value of uNK cells count in women with reproductive failure. Miscarriage rate uNK normal All cases Normal histological datinga Abnormal histological datingb uNK high All cases Normal histological datingc Abnormal histological datingd a vs b, p = 0.07. c vs d, p = 0.02.
44% (23/52) 36% (13/36) 62% (10/16) 41% (13/31) 36% (10/28) 100% (3/3)
work is also necessary to determine whether or not histological dating has any prognostic value on pregnancy outcome in the general infertility population. In our study, we used two separate methods (uNK cell count and histological dating) in endometrium in peri-implantation period. We think our study highlights an important concept in the evaluation of endometrium in women with reproductive failure. The vast majority of, if not all, previous studies focused on a single marker to assess endometrial function. However, our study suggests that multiple evaluations is necessary to improve the clinical usefulness of the measurements, just as multiple markers have been successfully developed to screen for ovarian cancer, prostate cancer, Down’s syndrome, etc. Our study appears to be the first of its kind to use combined measurements to prognosticate pregnancy outcome. The predictive value of the combined measurement may be further improved if the karyotype result of the abortus in women with recurrent miscarriage is known. If the karyotype result of the abortus is normal, the likelihood of an underlying endometrial abnormality is increased compared with the situation when the karyotype of the abortus is abnormal, as the endometrium in such a case is more likely to be normal. However, we do not have in our series karyotype result of the preceding miscarriage in the majority of subjects to enable us to carry out the additional analysis. It is desirable in future studies to incorporate fetal karyotyping results to verify to what extent the additional information improves the predictive value. One possible criticism of our study is that we included two categories of women with reproductive failure, namely, women with RM and RIF. Although they do have some differences, they both appear to have an increased prevalence of increased uNK cell count [19] and a presumed uterine factor to account for their reproductive failure. Among the 166 women included in the study, 94 women had RM and 72 women had RIF. Among 94 women with RM, 69 (73%) conceived within a year of the investigation and fulfilled the inclusion criteria whereas among 72 women with RIF, only 14 (19%) conceived within a year of the investigation and fulfilled the inclusion criteria. The apparent discrepancy in the conception rate (hence the inclusion) relates to the different reproductive performance of the two groups: RM subjects do not have problem conceiving whereas women with RIF do. Furthermore, women with recurrent miscarriage conceived spontaneously whereas women with RIF had to undergo further IVF treatment and received luteal support during early pregnancy. Nevertheless, if we had confined the analysis of the prognostic value of uNK cell and histological dating only to women with RM (n = 64), similar results were obtained: there was no significant correlation (p = 0.9) between pregnancy outcome and uNK cell count but there was a significant (p = 0.05) correlation with histological dating. It should be noted that there are two populations of uNK in the human endometrium. The majority (90%) are CD56+ CD16, but there is a small population (10%) of CD56+ CD16+ cells which resemble peripheral blood NK cells, and some studies have suggested that it is this specific population of cells that is associated with reproductive failure [21,28]. In this study, we only looked at the whole population of uNK cells. In a previous study, we have shown that the numbers of CD56+ cells, but not CD16+ cells, were increased in women with recurrent implantation failure after IVF [10] and this was the reason for concentrating on this cell population in this study. Previous studies have suggested that specific populations of endometrial T cells, Treg cells, may be important in recurrent miscarriage [29]. However, these cells only make up a very small proportion of the endometrial leucocyte population and they are a less obvious marker for predicting pregnancy loss than the CD56+ cell population.
B. Liu et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 181 (2014) 171–175
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