İmplantasyon ve Endometrium

... konulu sunumlar: "İmplantasyon ve Endometrium"— Sunum transkripti:

1 İmplantasyon ve Endometrium
Ömür Taskin Akdeniz Universitesi Tıp Fakültesi Kadın Hst. Doğ. Anabilimdalı

2 Apozisyon Adezyon Invazyon
implantasyon süreci : Apozisyon Adezyon Invazyon

3 Human embryo implantation in the uterus.
(A) Endometrium proliferates under estrogen enhancement. (B) Progesterone from luteinized follicles leads to endometrial differentiation. (C) The blastocystenters the uterus through the ostia and rolls freely over the endometrium under signals by L-selectin. (D) Mucin-1 (MUC-1) repels the blastocyst and prevents its adhesion to endometrial areas with poor chances of implantation. (E) Chemokines and cytokines attract the blastocyst to the optimal implantation spot. (F) Adhesion molecules (e.g. integrins and cadherins) firmly attach the blastocyst to the endometrial pinopods to ensure further successful implantation.

4 Figure 1. Epithelial cell adhesiveness by E-cadherin is controlled by intracellular calcium. Rising progesterone levels induce calcitonin expression and thus increase the concentration of intracellular calcium, which then suppresses E-cadherin expression at cellular contact sites.

5 Donor IVF

6 COH Endometrial ilerlemis maturasyon Erken pinopod oluşumu
Hızlanmış ER ve PR downregulasyonu Azalmış integrin oluşumu Artmış glycodelin A

7 menstruel siklusun :E ve P

8 Folliküler fazda tek gerekli olan; yeterli E2 desteğidir…

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13 E2 desteğinde gerekli olan miktarin değişebilirliği ayni zamanda E2 tedavisinin zamanini 10 gün ile 100 gün arasinda değiştirebilmektedir…

14 LUTEAL FAZ : ENDOMETRİAL GLANDLARDA VE STROMADA , ENDOMETRİAL DEĞİŞİKLİKLERİN KESİN BİR ŞEKİLDE ARDI ARDINA GELMESİ

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17 Mean value for natural cycle (± SD)
   TABLE: Sonographic characteristics of natural and letrozole cycles. Variable Mean value for natural cycle (± SD) Mean value for letrozole cycle (± SD) P value Ovulatory follicles 1.0 ± 0.0 2.0 ± 0.9 .02 Greatest follicle diameter (mm) 19.3 ± 2.1 23.8 ± 2.7 .01 Greatest endometrial thickness (mm) 12.1 ± 1.7 12.3 ± 2.3 .815 Ovulatory day 13.9 ± 2.7 14.0 ± 1.4 .199

18 TABLE: Endocrine profile of spontaneous and letrozole cycles.
Variable Mean value for natural cycle (± SD) Mean value for letrozole cycle (± SD) P value Day 7 FSH (IU/mL) 4.6 ± 0.7 4.9 ± 0.6 .63 Day 7 LH (IU/mL) 3.5 ± 0.4 5.9 ± 0.8 .003 Day 7 E2 (pmol/L) 161.5 ± 14.7 98.4 ± 11.4 .002 Preovulatory E2 (pmol/L) 427.2 ± 40.7 430.8 ± 48.8 .93 Preovulatory E2/mature follicle (pmol/L) 252.5 ± 49.9 .018 Day 7 A (nmol/L) 7.36 ± 1.36 8.58 ± 1.5 .016 Day 7 T (nmol/L) 1.28 ± 0.17 1.63 ± 0.21 .006 Postovulatory day + 7 Progesterone (pmol/L) 27.7 ± 4.6 44.2 ± 4.6 .008

19 FIGURE : Pinopode formation in endometria of control and letrozole-treated cycles evaluated
by scanning electron microscopy. (A) Control cycle endometrium (original magnification, ×1,500; bar = 10μm). (B) Letrozole-treated cycle endometrium of the same patient (original magnification, ×2,500; bar= 5 μm). (C) Control cycle endometrium (original magnification, ×3,000, bar = 5 μm). (D) Letrozole-treated cycle endometrium of the same patient (original magnification, ×1,500; bar = 10 μm).

20 Variable Natural cycles (n = 6) Letrozole cycles (n = 6) P value
TABLE : Endometrial dating and pinopode formation in natural and letrozole cycles. Variable Natural cycles (n = 6) Letrozole cycles (n = 6) P value Histological dating (mean ± SD) 18.6 ± 0.6 19.6 ± 0.6 0.506a In-phase biopsies (%) 3 (50) 6 (100) 0.182b Out-of-phase biopsies (%) 0 (0) NS Pinopode formation (SEM), positive samples (%) — Note: NS = not statistically significant. a Paired t test. b Fisher's exact test.

21 Estradiol düzeyi ve İmplantasyon?

22 E2’nin zararlı etkileri
Simon C. et al. Fertil steril 1998;70;234-9 86 high responders previous failed IVF >3 good quality embryos 24 step down 62 regular protocol STEP DOWN , hCG Step-dn Std P Age 31,6 33,9 NS Amps 22,4 E2 1919 5271 0,001 Oocytes 18,1 23,1 E.Trans. 3,3 3,4 E.Frozen 2,5 3,1 PR 64,2 24,2 <0,001 Impl R 29,3 8,5 0,02 OHSS 12,9 0,04

23 Yüksek E2 seviyesindeki endometriumun morfometrik analizi
Dogal siklus modarate COH E2<20,000pmol/l E2>20,000 pmol/L E2>20,000 pmol/L Yüksek cevap vericiler Gland- stroma arasındaki dissenkronizasyon: Gecikmiş glandular gelişme Ödemli stroma Basir GS et al. Human reprod. 2001;16:435-40

24 E2’nin Progesteron’a oranı:
dünün efsanelerinden bugünün gerçeklerine….

25 E2/ Progesteron endometrial morfoloji üzerindeki etkileri

26 E2/ Progesteron endometrial morfoloji üzerindeki etkileri

27 Pre-hCG P4 elevasyonu Zayif IVF sonuçlariyla iliskili olan P yükselmesi Schoolcraft W.Fertil Steril.1991;55:563-6 Mio Y. Fertil Steril. 1992;58:159-66 Dirnfeld M.J Assist Reprod Genet. 1993;10:126-9 Fanchin Fertil Steril. 1993;59:1090-4 Silverberg KM.J Clin Endocrinol Metab. 1991;73: IVF sonuçlarına etki etmeyen P yükselmesi Givens CR. Fertil Steril. 1994;62:1011-7 Hofmann GE. Fertil Steril.1996;66:980-6 Abuzeid MI. Fertil Steril. 1996;65:981-5 Lindheim SR. J Assist Reprod Genet. 1999;16:242-6

28 Pre-hCG P4 elevasyonu P>0.9 ng/ml P<0.9 ng/ml P
Zayif Orta kuvvetli P>0.9 ng/ml %3.20 %30 %34 P<0.9 ng/ml %23 %31 P <0.05 NS Fanchin R et al. Fertil Steril 1997;68: Fanchin ve arkadaslari, pre hCG P4 yükselmesinin pratik sonuçlarinin, COH’na olan cevabin derecesine bagli olduğunu bulmuslardir

29 COH’un sonuçlari: ‘üçüncü faktör’ hipotezi
COH: Hmg/fsh *Multiple follikuler gelisme için gerekli Foll. Phase Luteal I E2 II P4 III Peptides Androgens ?

30 3. Faktör Hipotezi

31 POTANSİYEL 3. FAKTÖRLER: TESTOSTERON
VE/VEYA DİĞER ANDROJENLERİ

32 Androjenler ve Reseptivite

33 Homebox genleri ve uterin/endometrial gelişim…

34 organogenesisi kodlayan faktörleri exprese eder
Homebox genleri organogenesisi kodlayan faktörleri exprese eder

35 Memeliler en az 39 HOX geni içerirler (hepsinin expresyon bölgeleri benzerdir).
Hoxa demetinden 4 tanesi reproduktif organlarda exprese olmaktadır; hoxa 9, hoxa 10, hoxa 11, hoxa 13.

36 Testosteron ve DHT, endometrium yeterli E2 ve progesterona maruz kaldığında hoxa 10 geninin expresyonunu bozmaktadır.

37 Sonuç olarak; testosteron/ DHT ‘da artma, hoxa 10 geninin expresyonunu azaltmaktadır ki, bu bulgu PCOS ‘da ve kontrollu ovarian hiperstimulasyonun belli formlarında görülmektedir (implantasyon oranları azalmakta ve düşükler artmaktadır).

38 Taskin et al, Fertil Steril P245,2004
ENDOMETRİAL RECEPTİVİTTY FOLLOWİNG EARLY START AND SİNGLE DOSE OF GnRH ANTOGONİST İN PATİENTS WİTH PCO Taskin et al, Fertil Steril P245,2004

39 Endometrial reseptivitenin biyolojik ve morfolojik markerları…

40 1.Endometrial morfolojik özellikler:
a.Histoloji b.pinopodlar

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42 2)Hücre adezyon molekül ailesi
a.İntegrinler b.Selektinler c.Kadherinler d.İmmunoglobulinler e.Musinler

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44 3.sitokinler LIF IL-6 IL-1 prostoglandinler

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50 Summary of studies performed in human endometrium using microarray analysis-1
Process studied Microarray Company Number of gene targets Study Decidualization Clontech Atlas array Stanford University     588 Popovici et al. (2000) Incyte human GEM-V Incyte Genomics     6918 Brar et al. (2001) Endometrial cancer Hu6800 Affymetrix     6000 Mutter et al. (2001) WOI HG-U95A 12 686 Kao et al. (2002) Carson et al. (2002) Endometriosis Human gene genefilter GF211 Research Genetics     4133 Eyster et al. (2002) Atlas human cDNA expression Array Clontech     597 Lebovic et al. (2002) Oncochip Centro Nacional de Investigaciones Oncológicas     6386 Moreno-Bueno et al. (2003a) Human cytokine expression array R&D Systems     375 Domínguez et al. (2003) HU-95A Tierney et al. (2003) Home-made University of Tokio 23 040 Arimoto et al. (2003) HG-U95A-E >60 000 Borthwick et al. (2003) Riesewijk et al. (2003) RU486 University of Cambridge  1000 Catalano et al. (2003) Kao et al. (2003) National Cancer Institute     9984 Risinger et al. (2003) Progesterone effect Human Chip 1K set 1 Takara Shuzo     1000 Okada et al. (2003)

51 Summary of studies performed in human endometrium using microarray analysis-2
Endometriosis Atlas human 1.2 cDNA expression array Clontech     1172 Matsuzaki et al. (2004) Endometrial cancer GEMarray clones Incyte Genomics 18 098 Cao et al. (2004) Home-made University of Cambridge     1056 Saidi et al. (2004) U133A Affymetrix >22 000 Ferguson et al. (2004) Stimulated cycles HG_U95Av2 12 686 Mirkin et al. (2004) Menstrual cycle Peter MacCallum Cancer Institute 10 500 Ponnampalam et al. (2004) Ferguson et al. (2005) WOI HG-U95Av2 Mirkin et al. (2005) Proliferative phase BD Atlas nylon cDNA expression array BD Biosciences Clontech     1200 Yanahiara et al. (2005) HG_U133A Horcajadas et al. (2005) Simón et al. (2005) HGU133 Plus 2.0 54 600 Talbi et al. (2005) HU-133A Punyadeera et al. (2005) RU486 >15 000 Sharkey et al. (2005) Matsuzaki et al. (2005) IUD >16 000 Horcajadas et al. (2006)

52 Different studies performed at the time of implantation in human using wide genomic analysis
Study Samples RNA pooled First sample (day of cycle) Second sample (day of cycle) Fold-change Up Down Kao et al. (2002) 11 No Proliferative phase (8–10) LH + (8–10) (21–23) >2.0 156 377 Carson et al. (2002) 6 YES LH + (2–4) (15–17) LH + (7–9) (20–22) 323 370 Borthwick et al. (2003) 10 Yes Proliferative phase (9–11) LH + (6–8) (19–21) 90 46 Riesewijk et al. (2003) LH + 2 (15) LH + 7 (20) >3.0 153 58 Mirkin et al. (2005) 8 Early-luteal (16) Mid-luteal (21) 49

53 Table :Genomic studies performed in COS cycles using GnRH agonists and
antagonists Treatment and direction of regulation Number of genes WOI genes Typically up-regulated (n = 894) Typically down-regulated (n = 504) Horcajadas et al. (2005) Leuprolide (agonist) Up 281 9 115 Down 277 227 Simón et al. (2005) Ganirelix 0.25 mg/day 22 4 69 46 Ganirelix 2 mg/day (antagonist) 88 7 24 15 1 Buserelin long protocol 3 100 76 2

54 Figure: Venn diagram comparing the three studies performed in our laboratory at the time of implantation in natural cycle (Horcajadas et al., 2005 ), in COS (Horcajadas et al., 2005 ) and in the presence of an inert IUD (Horcajadas et al., 2006 ) to find the consensus genes in endometrial receptivity.

55 Table: List of gene targets-1 Gene name Natural cycle COS IUD
Glutathione peroxidase 3 +25.87 –11.81 –13.40 Placental protein 14 +81.61 –9.83 –10.18 FXYD domain containing ion transport regulator 2 +4.07 –4.53 –9.43 Dipeptidylpeptidase 4 +31.37 –37.14 –8.47 Leukaemia inhibitory factor +36.62 –23.02 –4.57 Insulin-like growth factor-binding protein 3 +4.00 –4.26 –3.76 Growth arrest and DNA-damage-inducible, alpha +8.03 –3.03 –3.52 Hyaluronan-binding protein 2 +5.90 –6.41 –3.42 Endothelin receptor type B +8.21 –3.20 –3.41 Leiomodin 1 +29.73 –4.08 –3.10 Calponin 1, basic, smooth muscle +10.31 –9.26 –2.86 Clusterin +28.78 –7.13 –2.79 Transgelin +8.38 –3.72 –2.73 Cytochrome P450, family 2, subfamily C, polypeptide 9 +2.48 –4.66 –2.49

56 Tissue factor pathway inhibitor 2 Sorbitol dehydrogenase
Table: List of gene targets-2 Calpain 6 –4.58 +10.32 +11.20 Tissue factor pathway inhibitor 2 –3.52 +5.42 +7.32 Mitogen-activated protein kinase kinase 6 –4.87 +8.65 +6.57 Catenin, alpha 2 –8.44 +6.40 Sorbitol dehydrogenase –2.42 +11.56 +5.87 Major histocompatibility complex, class II, DO beta –16.48 +12.23 +4.93 Branched chain keto acid dehydrogenase E1, beta polypeptide –3.33 +4.06 Aldehyde dehydrogenase 3 family, member B2 –3.12 +4.28 +3.52 NDRG family member 2 –5.98 +5.90 +3.35 Folate receptor 1 –10.49 +9.30 +2.53 WOI genes that are dysregulated in COS cycles and in the presence of an IUD. Genes shown in bold did not recover their normal expression 1 year after IUD removal

57 Tedavi Secenekleri

58 Molecules Proposed mechanism Result
Intervention Proposed mechanism Result Progesterone (Nosarka et al., 2005) Vaginal/IM/oral Decreases uterine contractions Improves endometrial receptivity Two-fold increase in PR (meta-analysis) Hyaluronic acid (Simon et al., 2003) Embryos culture medium supplement Promotes cell–cell and cell–matrix adhesion No significant increase in PR or IR Heparin (Stern et al., 2003 Subcutaneous Inhibits binding of phospholipids with antibodies Protects the trophoblast from İnjury Aspirin (Rubinstein et al., 1999; Pakkila et al., 2005) Oral Increases endometrial blood flow and tissue perfusion Decreases uterine contractions Controversial results LIF (Brinsden et al., 2003) Prostaglandin E and TGF-ß (Tremellen et al., 2000) Intercourse around embryo transfer Semen components may induce embryo cleavage and immunotolerance No significant increase in PR Significant increase in IR IVIG (Coulam and Goodman, 2000; Stephenson and Fluker, 2000) Intravenous IgG administration Reduces detrimental natural killer (NK) cells activation

59 Methods used to treat conception failures
Molecules Intervention Proposed mechanism Result Progesterone (Nosarka et al., 2005) Vaginal/IM/oral Decreases uterine contractions Improves endometrial receptivity Two-fold increase in PR (meta-analysis) Hyaluronic acid (Simon et al., 2003) Embryos culture medium supplement Promotes cell–cell and cell–matrix adhesion No significant increase in PR or IR Heparin (Stern et al., 2003 Subcutaneous Inhibits binding of phospholipids with antibodies Protects the trophoblast from injury Aspirin (Rubinstein et al., 1999; Pakkila et al., 2005) Oral Increases endometrial blood flow and tissue perfusion Decreases uterine contractions Controversial results LIF (Brinsden et al., 2003) Improves endometrial receptivity Prostaglandin E and TGF-ß (Tremellen et al., 2000) Intercourse around embryo transfer Semen components may induce embryo cleavage and immunotolerance No significant increase in PR Significant increase in IR IVIG (Coulam and Goodman, 2000; Stephenson and Fluker, 2000) Intravenous IgG administration Reduces detrimental natural killer (NK) cells activation PR, pregnancy rates; IR, implantation rates; LIF, leukaemia inhibitory factor; TGF-ß, transforming growth factor-ß; IM, intramuscular; IVIG, intravenous IgG.

60 Endometrial reseptiviteyi arttırmada pratik yollar:
Bebek asprini kullanımı Kontrollu ovarian hipersitumaslyon ‘un geç evrelerinde FSH miktarını azaltarak Çok ince endometrium varlığında vaginal E2 uygulamak Antiandrogen kullanımı Follikular fazda ve muhtemel dexametazon kullanımında progesteron artışının saptanması