Basics

Scientific background


Endometrial biopsy


The endometrial biopsy can be obtained without anesthesia and, if indicated, may be combined with a diagnostic hysteroscopy. The endometrial biopsy is then preserved in formalin. As only little tissue is removed, the risks (injury, infection, bleeding) are very low.

In our experience patients respond differently to the biopsy: some feel no discomfort, while others feel a brief discomfort similar to the one experienced during period. Buscopan® and / or Voltaren® given 1 hour before the biopsy may help to relief. After the biopsy, the tissue is sent directly to Mannheim in our laboratory in special shipping material provided by us.


Uterine natural killer cells


Uterine natural killer cells are immune cells that play an important role in both the second half of the cycle (luteal phase) and in early pregnancy. They are called "killer" due to their ability to target viruses and bacteria as well as tumor cells. By contributing about 70% of immune cells in the first trimester of pregnancy at the feto-maternal interface, the uNK cells represent the most significant population of all immune cells (2).

Results of international studies on "uterine natural killer cells and pregnancy" show conflicting results (3). Overall however, an increasing body of evidence suggests that an elevated number of natural uterine killer cells may have a negative impact on implantation or pregnancy (1,4,5).

After analyzing more than 20 000 endometrial biopsies and conducting scientific studies on uNK cells, approximately 20-30% of women with recurrent implantation failure (RIF) or recurrent miscarriage (RSA) show an increase of uNK cells (6).


Uterine plasma cells


Uterine plasma cells accumulate in the endometrium in case of chronic endometritis.

Women with chronic endometritis are usually asymptomatic or show mild or nonspecific symptoms such as chronic pelvic pain, pain during sexual intercourse, irregular vaginal bleeding, and persistent vaginal discharge. This explains why it remains undiagnosed in many cases (7).

The surface marker CD138 is used to identify uterine plasma cells. Chronic endometritis is diagnosed in international studies when only one CD138-positive plasma cell is found, as well as when many plasma cells are found (8-10). This also explains why the incidence of chronic endometritis in women with RSA or RIF ranges from 9 to 57.8% in prior research (8,11-17).

According to our analysis of over 20 000 endometrial biopsies with a focus on chronic endometritis, the incidence among women with RIF and RSA is approximately 10-15%.




Uterine regulatory T-cells


Regulatory T-cells are T-lymphocytes that play an important role in immune system self-regulation by preventing an excessive immunological response, for example, against the body's own cells.

Uterine regulatory T cells regulate the activity of the immune system and can promote tolerance towards the embryo. These, like uterine natural killer cells, play a vital immunological role during implantation and the early stages of pregnancy.

The importance of regulatory T cells in the context of implantation and early pregnancy has been examined in various studies (18-21). Patients with RSA and RIF were found to have significantly less uterine regulatory T-cells in the endometrium than healthy controls (22-24). This indicates that a decrease in uterine regulatory T-cells may impair uterine receptivity (also known as endometrial receptivity) and embryo implantation.


Endometrial BCL6 expression


Background: What is BCL6?


B-cell lymphoma 6 (BCL6) is a nuclear protein used to diagnose B-cell lymphoma. BCL6 not only has crucial functions in cell cycle control, cell differentiation and inhibition of apoptotic

processes (25-26), it also stimulates the expression of pro-inflammatory cytokines (27-29). Chronic inflammation can result in implantation and bleeding disorders, as well as dysmenorrhea. Endometriosis is linked to inflammatory processes, increased endometrial proliferation, decreased apoptosis and altered cellular immunity. The estrogen dominance that is often present is also associated with a reduced response to progesterone (so-called progesterone resistance) (30-32).

Recently, a large number of publications on BCL6 expression in women with fertility disorders and/or endometriosis patients have been published (33-43). They were able to demonstrate that endometrial BCL6 overexpression can be detected more frequently in patients with endometriosis and that it has a progesterone-antagonistic effect on the endometrium. BCL6 overexpression impairs the endometrium throughout the implantation process and is thus suggested as a biomarker for a dysfunctional endometrium and a critical causal component for progesterone resistance.


How to analyse endometrial BCL6 expression?


The level of BCL6 expression in the endometrial glands can be determined semi-quantitatively using immunohistochemistry. The percentage of stained endometrial gland nuclei and staining intensity are assessed and used to calculate the so-called HSCORE. A BCL6 expression with an HSCORE > 1.4 is pathologic /abnormal (33, 34,37-40). The endometrial biopsy should be performed 7-10 days after ovulation in a natural cycle.


BCL6 Expression and fertility


There are numerous studies that focus on BCL6 expression in fertility patients with/without endometriosis (33-40). These results show a high sensitivity (93%) and specificity (96%) for the presence of endometriosis (39). In a prospective cohort study, 75% of patients with unexplained infertility had BCL6 overexpression in the endometrium, which was associated with a significantly decreased pregnancy and live birth rate (17.3 vs. 64.7% and 11.5 vs. 58.8%, respectively) (34). Furthermore, patients with RIF and RSA showed a significantly higher expression of BCL6 in the endometrium compared to healthy control women (35,37).



Therapeutic options


Treatment in case of increased natural killer cells


To date, there are no approved immunological therapies targeting natural killer cells in patients with recurrent miscarriage or recurrent implantation failure. Different treatment options are currently under investigation in international studies. These options include glucocorticoids (e.g., prednisolone), lipid infusions (e.g., Intralipid®) and immunoglobulins.

Most studies on the treatment of elevated uterine killer cells use glucocorticoids and lipid infusions, but at present the therapies represent a so-called “off label” use (19-21).

Glucocorticoids
In a study with women suffering from idiopathic recurrent miscarriages and elevated uterine killer cells, administration of 20 mg prednisolone orally daily from cycle day 1-21 resulted in a significant reduction in uterine killer cells (19). Nevertheless, it should be kept in mind that glucocorticoids may also cause side effects, such as the development of gestational diabetes, arterial hypertension, preterm labor, decreased birth weight and disorders of pediatric neurological development and cleft lip and palate (22-24).
Lipid infusions
Lipid infusions contain soybean oil and have been used for many years in the treatment of intensive care patients for nutritional therapy. Studies have shown that lipid infusions may lower the activity of natural killer cells in peripheral blood (25-28). A study focusing the administration of lipid infusions in women with elevated uterine killer cells does not yet exist so far. No side effects have been reported for the use of lipid infusions in the present studies in patients with implantation failure or miscarriage. However, an allergy to soy, peanuts and egg yolks must be ruled out before administration of soy-containing lipid infusions.


Treatment in case of elevated plasma cells or detection of chronic endometritis


Chronic endometritis is treated with antibiotics. Following this treatment, a decrease in the inflammatory response has been shown and is linked to an increased live birth rate (7,8,12). Among others, the broad-spectrum antibiotic doxycycline is usually recommended for 14 as first line therapy (12). Different doses are used in the studies (e.g. 100 mg doxycycline twice daily for 14 days (8,29)). Alternative therapy regimens may include antibiotics such as azithromycin, metronidazole, ciprofloxacin, levofloxacin, or amoxicillin/clavulanic acid.



Treatment in case of decreased numbers of uterine regulatory T-cells


It was demonstrated that the intrauterine administration of HCG led to an increase in uterine Tregs. Studies suggest that the administration of HCG to patients with RIF who show a reduction in uterine Tregs might be a possible new therapeutic approach (30, 31).

The new studies confirm the data that:

  • a subgroup of RM and RIF patients have significantly fewer Tregs in the endometrium, which may have a negative effect on endometrial receptivity
  • the intrauterine administration of HCG represents a possible therapeutic option (individual approach, off-label use) in order to increase the recruitment of uterine Tregs.


A potential approach is based on the results of a meta-analysis, which included the data of 15 randomized controlled studies with 2,763 patients, and recommends the administration of 500 IU HCG within 15 minutes before embryo transfer (32).



Treatment options in case of BCL6 overexpression


Likes et al. assessed the effect of pharmacological (GnRH agonist suppression for 2 months) or surgical (laparoscopy) therapy prior to embryo transfer in women with unexplained infertility and BCL6 overexpression in a prospective cohort study (38). The live birth rate was significantly higher in the treatment groups than in women who remained untreated. Also, during the ASRM Meeting 2020, further interim results on medical and surgical treatment based on data from 7 centers and 189 patients were presented, supporting the previously published data (44).


Your doctor will discuss potential therapy options with you, taking into account your personal history and individual risk factors.



Literatur

  1. Lash GE, Bulmer JN. Do uterine natural killer (uNK) cells contribute to female reproductive disorders? Journal of Reproductive Immunology. 2011 Mar;88(2):156–64.
  2. Kalkunte S, Chichester CO, Gotsch F, Sentman CL, Romero R, Sharma S. Evolution of non-cytotoxic uterine natural killer cells. Am J Reprod Immunol. 2008 May;59(5):425–32. 
  3. Seshadri S, Sunkara SK. Natural killer cells in female infertility and recurrent miscarriage: a systematic review and meta-analysis. Human Reproduction Update. 2014 May;20(3):429–38. 
  4. Lash GE, Bulmer JN, Innes BA, Drury JA, Robson SC, Quenby S. Prednisolone treatment reduces endometrial spiral artery development in women with recurrent miscarriage. Angiogenesis. 2011 Dec;14(4):523–32.
  5. Robson A, Harris LK, Innes BA, Lash GE, Aljunaidy MM, Aplin JD, et al. Uterine natural killer cells initiate spiral artery remodeling in human pregnancy. FASEB J. 2012 Dec;26(12):4876–85. 
  6. Kuon RJ, Weber M, Heger J, Santillán I, Vomstein K, Bär C, et al. Uterine natural killer cells in patients with idiopathic recurrent miscarriage. Am J Reprod Immunol. 2017 Jun 21;78(4). 
  7. Greenwood SM, Moran JJ. Chronic endometritis: morphologic and clinical observations. Obstet Gynecol. 1981 Aug;58(2):176–84. 
  8. McQueen DB, Perfetto CO, Hazard FK, Lathi RB. Pregnancy outcomes in women with chronic endometritis and recurrent pregnancy loss. Fertility and Sterility. 2015 Oct;104(4):927–31. 
  9. Johnston-MacAnanny EB, Hartnett J, Engmann LL, Nulsen JC, Sanders MM, Benadiva CA. Chronic endometritis is a frequent finding in women with recurrent implantation failure after in vitro fertilization. Fertility and Sterility. 2010 Feb;93(2):437–41. 
  10. 1 Kasius JC, Fatemi HM, Bourgain C, Sie-Go DMDS, Eijkemans RJC, Fauser BC, et al. The impact of chronic endometritis on reproductive outcome. Fertility and Sterility. 2011 Dec;96(6):1451–6. 
  11.  Bouet P-E, Hachem El 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. Fertility and Sterility. 2016 Jan;105(1):106–10. 
  12. Cicinelli E, Matteo M, Tinelli R, Lepera A, Alfonso R, Indraccolo U, et al. Prevalence of chronic endometritis in repeated unexplained implantation failure and the IVF success rate after antibiotic therapy. Human Reproduction. 2015 Feb;30(2):323–30. 
  13. Hachem El H, Crepaux V, May-Panloup P, Descamps P, Legendre G, Bouet P-E. Recurrent pregnancy loss: current perspectives. International Journal of Women's Health. Dove Press; 2017;Volume 9:331–45. 
  14. Kitaya K. Prevalence of chronic endometritis in recurrent miscarriages. Fertility and Sterility. 2011 Mar 1;95(3):1156–8. 
  15.  McQueen DB, Bernardi LA, Stephenson MD. Chronic endometritis in women with recurrent early pregnancy loss and/or fetal demise. Fertility and Sterility. 2014 Apr;101(4):1026–30. 
  16. Park HJ, Kim YS, Yoon TK, Lee WS. Chronic endometritis and infertility. Clin Exp Reprod Med. 2016;43(4):185. 
  17. Zolghadri J, Momtahan M, Aminian K, Ghaffarpasand F, Tavana Z. The value of hysteroscopy in diagnosis of chronic endometritis in patients with unexplained recurrent spontaneous abortion. Eur J Obstet Gynecol Reprod Biol. 2011 Apr;155(2):217–20. 
  18. Wang W-J, Liu F-J, Zhang X, Liu X-M, Qu Q-L, Li F-H, et al. Periodic elevation of regulatory T cells on the day of embryo transfer is associated with better in vitro fertilization outcome. Journal of Reproductive Immunology. Elsevier Ireland Ltd; 2017 Feb 1;119:49–53. 
  19. Liu S, Diao L, Huang C, Li Y, Zeng Y, Kwak-Kim JYH. The role of decidual immune cells on human pregnancy. Journal of Reproductive Immunology. Elsevier; 2017 Nov 1;124:44–53. 
  20. Kofod L, Lindhard A, Hviid TVF. Implications of uterine NK cells and regulatory T cells in the endometrium of infertile women. Hum Immunol. Elsevier; 2018 Sep 1;79(9):693–701. 
  21. Zhou J, Wang Z, Zhao X, Wang J, Sun H, Hu Y. An increase of Treg cells in the peripheral blood is associated with a better in vitro fertilization treatment outcome. Am J Reprod Immunol. 2012 Aug;68(2):100–6. 
  22. Jiang R, Yan G, Xing J, Wang Z, Liu Y, Wu H, et al. Abnormal ratio of CD57 +cells to CD56 +cells in women with recurrent implantation failure. Am J Reprod Immunol. 2017 May 20;78(5):e12708–8. 
  23. Bao S-H, Wang XP, De Lin Q, Wang W-J, Yin G-J, Qiu L-H. Decidual CD4+CD25+CD127dim/− regulatory T cells in patients with unexplained recurrent spontaneous miscarriage. European Journal of Obstetrics and Gynecology. Elsevier Ireland Ltd; 2011 Mar 1;155(1):94–8.
  24. Diao L-H, Li G-G, Zhu Y-C, Tu W-W, Huang C-Y, Lian R-C, et al. Human chorionic gonadotropin potentially affects pregnancy outcome in women with recurrent implantation failure by regulating the homing preference of regulatory T cells. Am J Reprod Immunol. 2017 Jan 3;77(3):e12618–8
  25. Kumagai, T. et al. The proto-oncogene Bcl6 inhibits apoptotic cell death in differentiation-induced mouse myogenic cells. Oncogene 18, 467–475 (1999).
  26. Kojima, S. et al. Testicular germ cell apoptosis in Bcl6-deficient mice. Development 128, 57–65 (2001).
  27. Yu, R. Y.-L. et al. BCL-6 negatively regulates macrophage proliferation by suppressing autocrine IL-6 production. Blood 105, 1777–1784 (2005). 
  28. Takeda, N. et al. Bcl6 Is a Transcriptional Repressor for the IL-18 Gene. J Immunol 171, 426–431 (2003).
  29. Chaouat, G., Dubanchet, S. & Ledée, N. Cytokines: Important for implantation? J Assist Reprod Gen 24, 491–505 (2007).
  30. Halis, G. & Arici, A. Endometriosis and Inflammation in Infertility. Ann Ny Acad Sci 1034, 300–315 (2004).
  31. Bulun, S. E. et al. Progesterone resistance in endometriosis: Link to failure to metabolize estradiol. Mol Cell Endocrinol 248, 94–103 (2006). 
  32. Young, S. & Lessey, B. Progesterone Function in Human Endometrium: Clinical Perspectives. Semin Reprod Med 28, 005–016 (2010). 
  33. Evans-Hoeker, E. et al. Endometrial BCL6 Overexpression in Eutopic Endometrium of Women With Endometriosis. Reprod Sci 23, 1234–1241 (2016). 
  34. Almquist, L. D. et al. Endometrial BCL6 testing for the prediction of in vitro fertilization outcomes: a cohort study. Fertil Steril 108, 1063–1069 (2017). 
  35. Gong, Q. et al. Increased levels of CCR7(lo)PD-1(hi) CXCR5+ CD4+ T cells, and associated factors Bcl-6, CXCR5, IL-21 and IL-6 contribute to repeated implantation failure. Exp Ther Med 14, 5931–5941 (2017). 
  36. Yoo, J.-Y. et al. KRAS Activation and over-expression of SIRT1/BCL6 Contributes to the Pathogenesis of Endometriosis and Progesterone Resistance. Sci Rep-uk 7, 6765 (2017). 
  37. Fox, C. W. et al. Unexplained recurrent pregnancy loss and unexplained infertility: twins in disguise. Hum Reproduction Open (2019) doi:10.1093/hropen/hoz021. 
  38. Likes, C. E. et al. Medical or surgical treatment before embryo transfer improves outcomes in women with abnormal endometrial BCL6 expression. J Assist Reprod Gen 36, 483–490 (2019). 
  39. Nezhat, C. et al. BCL-6 Overexpression as a Predictor for Endometriosis in Patients Undergoing In Vitro Fertilization. Jsls J Soc Laparosc Robotic Surg 24, e2020.00064 (2020). 
  40. Klimczak AM, Herlihy NS, Scott CS, Hanson BM, Kim JG, Titus S, Seli E, Scott RT Jr. B-cell lymphoma 6 expression is not associated with live birth in a normal responder in vitro fertilization population. Fertil Steril. Feb;117(2):351-358 (2022). 
  41. Kosturakis, A. K. & Ryan, G. L. Predicting implantation failure: to BCL6 or not to BCL6. Fertil Steril 117, 359 (2022). 
  42. Lessey, B. A. & Kim, J. J. Endometrial receptivity in the eutopic endometrium of women with endometriosis: it is affected, and let me show you why. Fertil Steril 108, 19–27 (2017). 
  43. Sansone, A. M. et al. Evaluation of BCL6 and SIRT1 as Non-Invasive Diagnostic Markers of Endometriosis. Curr Issues Mol Biol 43, 1350–1360 (2021). 
  44. Dan, A., Outcomes in women with IVF Failure who tested positive for BCL6 using ReceptivaDxTM Testing: effect of treatment on subsequent embryo transfer. Fertil Steril 113(4):E13 (2020)
  45. Quenby S, Kalumbi C, Bates M, Farquharson R, Vince G. Prednisolone reduces preconceptual endometrial natural killer cells in women with recurrent miscarriage. Fertility and Sterility. 2005 Oct;84(4):980–4. 
  46. Tang A-W, Alfirevic Z, Turner MA, Drury JA, Small R, Quenby S. A feasibility trial of screening women with idiopathic recurrent miscarriage for high uterine natural killer cell density and randomizing to prednisolone or placebo when pregnant. Human Reproduction. 2013 Jul;28(7):1743–52. 
  47. Kuon RJ, Müller F, Vomstein K, Weber M, Hudalla H, Rösner S, et al. Pre-Pregnancy Levels of Peripheral Natural Killer Cells as Markers for Immunomodulatory Treatment in Patients with Recurrent Miscarriage. Arch Immunol Ther Exp (Warsz). 2017 Mar 11. 
  48. Lédée N, Vasseur C, Petitbarat M, Chevrier L, Vezmar K, Dray G, et al. Intralipid® may represent a new hope for patients with reproductive failures and simultaneously an over-immune endometrial activation. Journal of Reproductive Immunology. 2018 Nov;130:18–22. 
  49. Park-Wyllie L, Mazzotta P, Pastuszak A. Birth defects after maternal exposure to corticosteroids: prospective cohort study and meta-analysis of epidemiological studies. Teratology. 2000;62(6):385–92. 
  50. Hasbargen U, Reber D, Versmold H, Schulze A. Growth and development of children to 4 years of age after repeated antenatal steroid administration. Eur J Pediatr. 2001 Sep;160(9):552–5. 
  51. Laskin CA, Bombardier C, Hannah ME, Mandel FP, Ritchie JW, Farewell V, et al. Prednisone and aspirin in women with autoantibodies and unexplained recurrent fetal loss. N Engl J Med. 1997 Jul 17;337(3):148–53. 
  52. Granato D, Blum S, Rössle C, Le Boucher J, Malnoë A, Dutot G. Effects of parenteral lipid emulsions with different fatty acid composition on immune cell functions in vitro. JPEN J Parenter Enteral Nutr. 2000 Mar;24(2):113–8. 
  53. Roussev RG, Ng SC, Coulam CB. Natural killer cell functional activity suppression by intravenous immunoglobulin, intralipid and soluble human leukocyte antigen-G. Am J Reprod Immunol. 2007 Apr;57(4):262–9. 
  54. Roussev RG, Acacio B, Ng SC, Coulam CB. Duration of intralipid“s suppressive effect on NK cell”s functional activity. Am J Reprod Immunol. 2008 Sep;60(3):258–63. 
  55. Coulam CB, Acacio B. Does Immunotherapy for Treatment of Reproductive Failure Enhance Live Births? Am J Reprod Immunol. 2012 Feb 16;67(4):296–304. 
  56. Lee SK, Kim JY, Han AR, Hur SE, Kim CJ, Kim TH, et al. Intravenous Immunoglobulin G Improves Pregnancy Outcome in Women with Recurrent Pregnancy Losses with Cellular Immune Abnormalities. Am J Reprod Immunol. John Wiley & Sons, Ltd (10.1111); 2016 Jan;75(1):59–68. 
  57. Cohen BM, Machupalli S. Use of Gammaglobulin to Lower Elevated Natural Killer Cells in Patients with Recurrent Miscarriage. J Reprod Med. 2015 Jul;60(7-8):294–300. 
  58. Ramos-Medina R, García-Segovia A, Gil J, Carbone J, Aguarón de la Cruz A, Seyfferth A, et al. Experience in IVIg therapy for selected women with recurrent reproductive failure and NK cell expansion. Am J Reprod Immunol. 2014 May;71(5):458–66. 
  59. Kitaya K, Matsubayashi H, Takaya Y, Nishiyama R, Yamaguchi K, Takeuchi T, et al. Live birth rate following oral antibiotic treatment for chronic endometritis in infertile women with repeated implantation failure. Am J Reprod Immunol. 2017 Jun 13;78(5):e12719–8. 
  60. Vitagliano A, Saccardi C, Noventa M, Di Spiezio Sardo A, Saccone G, Cicinelli E, et al. Effects of chronic endometritis therapy on in vitro fertilization outcome in women with repeated implantation failure: a systematic review and meta-analysis. Fertility and Sterility. 2018 Jul 1;110(1):103–112.e1. 
  61. Schumacher A, Brachwitz N, Sohr S, Engeland K, Langwisch S, Dolaptchieva M, et al. Human Chorionic Gonadotropin Attracts Regulatory T Cells into the Fetal-Maternal Interface during Early Human Pregnancy. J Immunol. 2009 Apr 20;182(9):5488–97. 
  62. Tsampalas M, Gridelet V, Berndt S, Foidart J-M, Geenen V, Hauterive SPD™. Human chorionic gonadotropin: A hormone with immunological and angiogenic properties. Journal of Reproductive Immunology. Elsevier Ireland Ltd; 2010 May 1;85(1):93–8. 
  63. Wirleitner B, Schuff M, Vanderzwalmen P, Stecher A, Okhowat J, Hradecký L, et al. Intrauterine administration of human chorionic gonadotropin does not improve pregnancy and life birth rates independently of blastocyst quality: a randomised prospective study. Reprod Biol Endocrinol. Reproductive Biology and Endocrinology; 2015 Jul 3;:1–10. 
  64. Gao M, Jiang X, Li B, Li L, Duan M, Zhang X, Tian J, Qi K. Intrauterine injection of human chorionic gonadotropin before embryo transfer can improve in vitro fertilization-embryo transfer outcomes: a meta-analysis of randomized controlled trials. Fertil Steril. 2019 Jul;112(1):89-97.e1. 
  65. Zhang T, Chen X, Wang CC, Li TC, Kwak-Kim J. Intrauterine infusion of human chorionic gonadotropin before embryo transfer in IVF/ET cycle: The critical review. Am J Reprod Immunol. 2019 Mar 25;81(2):e13077–12. 
  66. Osman A, Pundir J, Elsherbini M, Dave S, El-Toukhy T, Khalaf Y. The effect of intrauterine HCG injection on IVF outcome: a systematic review and meta-analysis. Reprod Biomed Online. Elsevier Ltd; 2016 Sep 1;33(3):350–9. 
  67. Fox C, Azores-Gococo D, Swart L, Holoch K, Savaris RF, Likes CE, Miller PB, Forstein DA, Lessey BA. Luteal phase HCG support for unexplained recurrent pregnancy loss - a low hanging fruit? Reprod Biomed Online. 2017 Mar;34(3):319-324. 


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