Ovaryen yaşlanmada moleküler belirteçler Prof.Dr.Tülay İrez Biruni Üniversitesi Tıp Fakültesi Histoloji ve Embriyoloji Anabilim Dalı

Kadın infertilitesi insidansında artış Between years 1970 & 2002, the incidence of first child birth in women over 30 years of age increased by six folds % Fecundability  with  age Kadın infertilitesi insidansında artış Crawford NM, et al. Obstet Gynecol Clin North Am. 2015; 42(1): 15-25.

Üreme yeteneğinde düşme YAŞLANMA; Üreme yeteneğinde düşme Diğer dokulara kıyasla ovaryumda daha belirgin olarak ortaya çıkmaktadır Templeton A, Morris J K, Parslow W. Factors that affect outcome of in vitro fertilisation treatment. Lancet, 1996, 348: 1402–1406. Baird D T, Collins J, Egozcue J, et al. Fertility and ageing. Hum Reprod Update, 2005, 11: 261–276

Folikül rezervi yaşa bağlı olarak azalmaktadır Ericson 1986

Ovaryum yaşlanması A. Foliküllerin kalite ve sayısı, folikülogenez B. Ovarian yaşlanmada olaylar C. Ovaryen Yaşlanma belirteçleri Ovaryum yaşlanması

Correlation between POR and outcome of pregnancy  embryo quality with advancing age  spontaneous fecundity with advancing age  oocyte numbers  embryo numbers  pregnancy rates with infertility treatments in general  aneuploidy with advancing age POR leads to poor outcome of pregnancy mainly because of two factors- Decreased level of intra-ovarian androgens Oxidative stress due to Reactive Oxidative Species (ROS) Faddy MJ. Mol Cell Endocrinol. 2000; 163(1-2): 43-8/ Oudendijk JF, et al. Hum Reprod Update. 2012; 18(1): 1-11/ Gliecher N, et al. Reproductive Biology and Endocrinology. 2011, 9:23.

Figure 2 Schematic representation of follicle development emphasizing that AMH is produced in early stages of follicle development (characterized by gonadotrophin-independent growth), as opposed to Inhibin B and estradiol produced by follicles at later stages of development where growth is FSH-dependent. Adapted from McGee and Hsueh (2000). From: Anti-Müllerian hormone: ovarian reserve testing and its potential clinical implications Hum Reprod Update. 2014;20(5):688-701. doi:10.1093/humupd/dmu020 Hum Reprod Update | © The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com 8

Holte J, Brodin T, Berglund L, Hadziosmanovic N, Olovsson M, Bergh T Fertil Steril. 2011 Sep;96(3):594-9. doi: 10.1016/j.fertnstert.2011.06.071. Antral follicle counts are strongly associated with live-birth rates after assisted reproduction, with superior treatment outcome in women with polycystic ovaries

Ovaryen yaşlanmanın endokrinolojisi A. Menstrual siklus B. Siklus düzeninde bozulma C. Nöroendokrin değişiklikler D. Değişen ovaryum feed-back E. monotropik FSH yükselmesi F. Folikül fonksiyonu ve oosit yeterliliğinde azalma Ovaryen yaşlanmanın endokrinolojisi

Ovaryen rezerv belirteçleri      1 - yaş      2 - Bazal serum FSH değeri     3 - Bazal serum estradiol      4 - Bazal LH/FSH oranı      5 - Bazal serum inhibin-B seviyesi      6 - Bazal serum anti-Müllerian hormone düzeyi      7 - Bazal ovarian volüm      8 - Bazal antral follicle sayımı      9 - Ovarian stromal kan akımı    

Zayıf ovaryen rezerv nedir? Aynı zamanda prematür ovaryen yaşlanma da denir(POR) ~ 10% kadın, yaşa spesifik standard dışındadır ölçüm Normal POR FSH ≤ 12 IU/ml > 12 IU/ ml AMH 2-4 ng/ ml < 1 ng/ml AFC ≥ 10 < 5

Results: POR definition Following the same logical approach utilized for polycystic ovarian syndrome (PCOS) diagnostic criteria (The Rotterdam ESHRE/American Society for Reproductive Medicine (ASRM) Sponsored PCOS Consensus Workshop Group, 2004), a consensus was reached on the minimal criteria needed to define POR. At least two of the following three features must be present: (i) Advanced maternal age (≥40 years) or any other risk factor for POR; (ii) A previous POR (≤3 oocytes with a conventional stimulation protocol); (iii) An abnormal ovarian reserve test (i.e. AFC ,5–7 follicles or AMH ,0.5 –1.1 ng/ml). ESHRE sonuçları

Poor ovarian response (POR) - ART In the UK, IVF cycles involving women aged 40 years accounted for 9.2% of cycles in 1991, 10.7% in 2000, 15.5% in 2006 HFEA,2011 196 IVF centers in 45 countries reported an increase in the burden of POR over the last decade 25.8% of IVF clinicians surveyed stated that they add DHEA as an adjuvant to IVF treatment protocols in women with POR IVF Worldwide Survey, 2010

Ovaryen rezerv ölçümü FSH (Folikül Stimulan Hormon) - Düşük düzeyi daha iyi (Normal <10 iu/L) siklus 2-4. günlerde bakılması gerekli Over rezervi düşük olduğunda siklusta dalgalanma gösterir. AMH (Anti Müllerian Hormon) Yüksek olması daha iyi (normal>5pmol/L) Siklusda değişkenlik göstermez

FSH düzeylerinde ovarian yanıt <10 normal 10-12 sınırda 13-15 yükselen FSH, azalmış over rezervi, azalmış canlı doğum oranı 16-20 yükselmiş FSH, düşük embriyo sayı ve kalitesi >20 çok zayıf, stimulasyona yanıt yok FSH düzeylerinde ovarian yanıt

yaş unite Normal değer 24 aydan daha önce ng/mL pmol/l < 5 < 35 24 ay- 12 yaş < 10 < 70 13–45 yaş 1 - 10 7 - 70 > 45 yaş < 1 < 7

Serum estradiol Bazal FSH normal olduğunda bile yükseltilmiş bazal östradiol zayıf cevabı öngörebilir. Yumurtalık rezervinin tahmininde siklusun 3. günündeki östradiol düzeyinin değeri hala tartışmalıdır. Inhibin B Inhibin-B esas olarak büyüyen foliküllerde granülosa hücreler tarafından üretilir ve diğer serum testlerine göre yumurtalık aktivitesinin daha derhal değerlendirilmesini sağlar. 3. günde inhibitin-B düzeylerinde bir düşüş, 3. FSH gününde beklenen artıştan önce kötü yumurtalık rezervini öngörebilir.

AMH düzeyini etkileyen faktörler nelerdir? A. AMH düzeyini azaltan faktörler • ileri yaş • Obesite • Gonadotropin uygulaması • kemoterapi, radyoterapi • over cerrahisi B. AMH düzeyini artıran faktörler • Polikistik Ovarian Sendromu AMH düzeyini etkilemeyen faktörler • siklus günü • GnRH agonistleri • doğum kontrol ilaçları • gebelik C.AMH düzeyini etkilemeyen faktörler Normal ve anormal AMH düzeyleri nedir? • AMH düzeyi 0.2 - 0.5 ng/mL arasında ise daha az yumurta eldesi ve siklus iptali riski • AMH düzeyi 2.5 ng/mL üzerinde ise daha fazla sayıda yumurta eldesi ve daha iyi fertilite potansiyeli.

From: Ovarian Aging: Mechanisms and Clinical Consequences Fig. 7 Individual AMH levels (log scale) in relation to age in regularly menstruating fertile controls (regular cycles, FSH <10 U/liter) and three subgroups of patients with elevated FSH levels: 1) imminent ovarian failure (IOF, regular cycles, FSH >12 U/liter); 2) transitional ovarian failure (TOF, irregular cycles >35 d, with FSH >12 IU/liter without fulfilling the POF criteria); and 3) POF (at least one episode of secondary amenorrhea of ≥4 months, FSH >40 IU/liter). The data show that AMH level may be a preferred marker for the diagnosis of POF. In contrast to patients with IOF and TOF, where a considerable overlap exists, POF patients show undetectable AMH levels in almost 100% of cases. In TOF patients, undetectable AMH may be predictive of the expected period until the final menstrual bleeding (65). Reprinted with permission from (109). Solid lines, P5 AMH serum level for age; ++++, the menopausal threshold value for AMH (arbitrarily set at 0.086 μg/ml); and dashed lines, the lower level of detection of the AMH assay (0.023 μg/ml). Reprinted with permission from Ref. 109 . From: Ovarian Aging: Mechanisms and Clinical Consequences Endocr Rev. 2009;30(5):465-493. doi:10.1210/er.2009-0006 Endocr Rev | Copyright © 2009 by The Endocrine Society 21

AMH tip II reseptör mutasyonu AMHR2 mutasyonu ile doğuma bağlı menopoz yaşı arasında bir ilişki ? AMHR2, TGF-β ailesinden bir protein İnisyal folikül toplanmasında önemli rolü var Nullipar ve AMHR2 için genotipi (rs2002555) G/G olan kadınlar, A/A olanlardan 2.6 yıl erken menopoza girmektedirler. AMH tip II reseptör mutasyonu (Kevenaar ME, Hum Reprod 22:2382, 2007, Voorhuis M, (J Clin Endocrinol Metab 96: E473–E479, 2011)

Antral Folikül sayımı (AFC) Yüksek olması iyi 5-10 AF/ ovaryum –normal rezerv <3 AF / ovaryum –zayıf rezerv >10-15 AF / ovaryum – ‘polikistik’ Menstrual siklus uzunluğu – Kısalma döngüleri, yumurtalık rezervinin bozulduğunu gösterir.

Genetik Ovaryen Yaşlanma Nedenleri Michel De Vos, Paul Devroey, Bart C J M Fauser, 2010

Michel De Vos, Paul Devroey, Bart C J M Fauser, 2010

POF X-kromozom delesyonları Komplet Xp11 Xq13 Xq22 Parsiyel Xp21 Xp22 Xq26 Xq28

Over ve folikül içindeki mikroçevredeki enerji homeostasisinin sağlanması matür ve işlevsel tam bir oositin meydana gelmesini sağlar. (Tatone C and Amicarelli F. Fertil Steril 2013;99:12–7)

Ovaryum mikroçevresi ve oosit maturasyonuna etkileri 1. AGE’ler 2.Mitokondrial yetersizlikler 3.Stromal diferansiyasyon eksiklikleri 4.Mayoz hataları 5.AMH azalması 6.Teka hücre tabakasında yetersizlik,yetersiz androjen üretimi 7. Telomer kısalması ,telomeraz aktivitesi azalması

Genetik Faktörler Reaktif oksidatif stres (ROS) Metabolizma sonucu oluşan yüksek reaktivasyona sahip serbest radikallerdir. Yaşlanma ile birlikte hücresel solunumun düşmesi ile birlikte intramitokondrial elektron kaçağı artar ROS oluşumu artar. ROS antioksidanların karşılama kapasitesini geçerse, oksidatif ürünler mitokondrilerden sitokrom c ve diğer apoptozis tetikleyici faktörleri salarak hücre ölümüne sebep verir. AGEs (advanced glycation end products) AGEs formasyonu yaşlanmayı, aterosklerosis, DM, hızlandıran geriye dönüşümsüz bir proses. RAGE reseptörleri var ve intrasellüler OS ürünlerini arttırmaktadır Genetik Faktörler

Yaşlanma ile ilgili fiziksel & kimyasal stimulasyonlar Reaktif oksijen türevleri - Oosit hasarı Yaşlanma ile ilgili fiziksel & kimyasal stimulasyonlar Folikül atrezisi (apoptosis) Folikül sayısı  Ovarian Rezervi  Ovaryum ROS Oksidatif Stres  hızlanmış Oosit yaşlanması & Oosit kalitesi  ROS Ovulasyon süreci Mitokondrial aktivite  ATP sentezi  Antioksidan enzim aktivitesi  Kromozomal hasar  ROS are produced within follicle, especially during ovulatory process ROS play physiological role in process of ovulation, e.g. follicle rupture. However, excessive amount of ROS cause OS & may damage oocyte and granulosa cells ROS accelerate oocyte aging & deteriorate oocyte quality Infertilite Abortus DNA anormallikleri Jinno M, et al. Anti-Aging Medicine. 2012; 9 (1) : 6-13.

Genetik Faktörler Mitokondrial DNA (mtDNA) mutasyonları Foliküler ve germ hücrelerindeki maternal mtDNA hücre enerji homeostasisinden sorumlu olup , hücre bölünme ve apoptosisini düzenler Genetik Faktörler

Mitokondrial tedavi seçeneği

(TTAGGG)(n) repeats Telomeraz aktivitesindeki azalma Telomeraz, kromozomların sonlarında bulunan tekrarlayan DNA sekanslarıdır. Kromozomların uçlarında bir koruyucu başlık oluşturarak, kormozomların uç-uca füzyonlarını önler. Telomeraz her hücre bölünmesi ile kısalır ve neticede hücre bölünmesi arest olur, apoptozis oluşur ve genomik instabilizasyon olur. Reprod Fertil Dev. 2009;21(1):10-4. Telomeres and reproductive aging. Keefe DL, Liu L

Telomer ve telomerase (preliminer çalışma) İnfertilite tipi Telomerase + Telomerase - % DOR (7) 1 6 86 MF (16) 4 12 %75 TF (9) 3 %33 X square for trend 0,503 P=0,025 Telomer ve telomerase (preliminer çalışma)

LUF Sendromu Somatik hücrelerde yüksek mtDNA delesyon oranı Anormal mtDNA lı oositlerde, normalde ATP yapımını uyaran Ca+2, uyarımını yapamaz : spindle formasyonu, normal kromozomal dizilim ?????? LUF Sendromu (Seifer D B, Fertil Steril, 78: 1046, 2002, Gordo A C, Biol Reprod, 66: 1828, 2002, Eichenlaub-Ritter U, RBO, 8: 45, 2004 )

Composite micrographs of confocal images of the meiotic spindles in oocytes obtained from naturally cycling women in two age groups, 20–25 yr (top) and 40–45 yr (bottom). Chromatin is represented in red (propidium iodide) and microtubules in green (fluorescein isothiocyanate). Top, Oocytes obtained from the younger age group at metaphase II exhibit a uniform matrix of microtubules in the spindle and a distinct, uniform chromosome distribution within the metaphase plate. Bottom, Oocytes obtained from the older age group with microtubules in disarray compared with the oocytes in the top panels. A distinct metaphase plate of chromosomes is not visible because most chromosomes were not well aligned or dislocated from the metaphase plate. Doğal siklusta mayoz iğ ipliklerinin yaşa bağlı olarak kromozom dağılımına etkileri vardır

Foliküler büyüme oosit ve granulosa hücrelerinde yeterlilik ile sonuçlanır

Foliküler atreziye en fazla duyarlı aşama Tekal tabaka oluşumu: Foliküler atreziye en fazla duyarlı aşama Theca hücreleri olgunlaşır ve LH kontrolü altında steroidojen olurlar. üretilen androjenler miktarı estradiole dönüştürülür Theca hücreleri seçilir ve farklılaşır; LH reseptörleri, steroidojenik enzimler & az miktarda androjen üretirler Theca hücreleri luteinize olur Geçici endokrin bez Progesteron üretmek için işlev değiştirir Foliküllerden gelen sinyaller teka hücrelerinin oluşumunu sağlar Teka yok Steroid üretimi yok Primer Primodial The LH surge is a signal from the brain to the ovary that an egg is mature. The brain senses the elevated estrogen levels produced by the maturing egg and releases a burst of luteinizing hormone (LH). When the ovary is bombarded with this burst of LH, the sac-like covering (called the follicle) surrounding the egg thins, allowing the egg to escape. Folikülogenez Preantral korpus luteum Antral Young JM, et al. Reproduction. 2010; 140(4): 489-504.

Folikül-oosit Kompleksinin Genetik kontrolü (Mouse model) Genetic dissection of female fertility pathways in mice. An update of the figure from our review six years ago1 shows a marked increase in the gene products that have key roles at various states of ovarian folliculogenesis and after ovulation (the newly identified genes since the last review are in blue). Women have a pool of resting oocytes in the form of primordial follicles. Once these follicles are depleted, a woman can no longer have children naturally. Several master oocyte-specific transcriptional regulators interact to control primordial follicle formation and follicle maintenance and recruitment into the growing pool, as indicated. At later steps in folliculogenesis and through ovulation, paracrine factors (for example, KIT ligand, GDF9 and BMP15), autocrine factors (for example, activins and inhibins) and endocrine hormones (for example, FSH, LH, estradiol and progesterone) play key parts. After ovulation, proteins of the zona pellucida and oocyte maternal factors permit proper fertilization and the substantial changes in gene expression necessary for early embryogenesis. Ovarian prostaglandins and steroids are also essential to initiate a cascade of events in the uterus that readies it for implantation of a healthy embryo. Multiple proteins and factors are required for placentation and maternal behavior. Matzuk & Lamb, 2008

Primordialden primer foliküle geçiş Fare modellerine bağlı olarak, folliculogenesise özgü basic helix-loop-helix (FIGLA) doğumda kritik olan oocyte-spesifik transcription faktör olarak önem kazanmakta. Bu proteinin yokluğunda , yenidoğanda oositler yaşamaz, sonuç olarak steriliteye neden olur Forkhead box L2 (Foxl2) bir transcription faktorüdür ve pre-granulosa hücrelerinde eksprese edilir. Somatik hücrelerin oositlerin etrafındaki granulosa hücrelerine dönüşümünü sağlar ve primordial foliküllerin oluşması gerçekleşir. POF ‘lu kadınlarda FIGLA and FOXL2 mutasyonlarından söz edilir, bu proteinlerin insan folikülogenesisinde önemi olduğu ileri sürülmektedir

‘Bone Morphogenetic Protein 15 Gene’ (BMP15) Folikül gelişiminde rol oynayan ‘oocyte-derived growth and differentiation factor’ kodlar BMP15 gene (MIM *300247) encodes for an oocyte-derived growth and differentiation factor which is involved in 359 follicular development as a critical regulator of many GC processes (Dube et al. 1998; Chang et al. 2002; McNatty et 360 al. 2004; Shimasaki et al. 2004). As other TGF-β, BMP15 gene encodes a pre-proprotein consisting of a signal peptidea pro-region and a mature domain that can form homo- or hetero-dimers with related factors, such 361 as GDF9 (Chang et 362 al. 2002) (Figure 2). The BMP15 main roles include: a) the promotion of follicle maturation since the primordial 363 gonadotrophin-independent phases of folliculogenesis; b) regulation of follicular GC sensitivity to FSH action; c) 364 prevention of GC apoptosis; d) promotion of oocyte developmental competence; e) regulation of ovulation quota 365 (Shimasaki et al. 2004; Fabre et al. 2006; Hussein et al. 2005; Hussein et al. 2006; Hashimoto et al. 2005). The 366 relevance of BMP15 action in ovarian folliculogenesis was initially shown by experimental and natural models. All 367 together, the data so far collected in different mammalian species indicate that the role of BMP15 may be more critical 368 in mono-ovulating species (such as sheep and human) than in the poli-ovulating ones (mice). Experimental disruption of 369 Bmp15 gene in mice cause a mild fertility defect limited to females (Yan et al. 2001), whereas natural missense 370 mutations in several strains of ewes cause a hyperprolificacy phenotype in the heterozygous state (increased litter size to 371 3-5 lambs per litter against 1 in wild-type) and a female infertility with complete block of folliculogenesis in the 372 homozygous state (FecX factors; Galloway et al. 2000; Davis et al. 2004; Hanrahan et al. 2004; McNatty et al. 2005; 373 Bodin et al. 2007). BMP15 maps to a locus on the short arm of X chromosome (Xp11.2) within a “POF critical region” 374 where are located several of the Turner syndrome traits including ovarian failure (Zinn et al. 1998; Persani et al. 2009). 375 In humans, mutations in BMP15 gene have been found in association with both primary and secondary amenorrhea in 376 several worldwide POI cohorts with a variable prevalence between 1.5-12% (Table 2). The first heterozygous mutation 377 of BMP15 gene (p.Y235C) was reported by us in 2 Italian sisters with hypergonadotropic ovarian failure characterized 378 by primary amenorrhea and ovarian dysgenesis, who inherited the genetic alteration from the unaffected father (Di 379 Pasquale et al. 2004). This mutation was located in a residue highly conserved among species and generated aberrant 380 high molecular weight products in vitro as seen at western-blot in non-reducing conditions, a likely consequence of the 381 additional Cys in the pro-region. A bioassay on primary cultures of human granulosa cells showed an impairment of the 382 growth stimulatory activity of recombinant mutant BMP15 in comparison with wild-type protein. Co-incubation 383 experiments of wild-type and p.Y235C proteins were consistent with a DNE exerted by mutant on the stimulatory 384 activity of wild-type protein on granulosa cells (Di Pasquale et al. 2004) (Figure 3). Afterwards, other variants have 385 been identified with variable frequency in patients from Europe, USA, North Africa, India, China and Asia (Di Pasquale 386 et al. 2006; Laissue et al. 2006; Dixit et al. 2006; Rossetti et al. 2009; Wang et al. 2010; Tiotiu et al. 2010) (Figure 4 387 and Table 2). Almost all of these are missense variations found in the heterozygous state. These variations are also 388 located in the gene sequence encoding the pro-region of the protein. More recently, we studied the recombinant 389 products of several of these missense variations and showed an impaired amount of mature BMP15 protein produced by 390 variant vectors in comparison with wild-type, suggesting a hampered processing. Consistently, we showed a significant 391 reduction of their biological effects by using a novel BMP-responsive luciferase-reporter assay on a human granulosacell line (Rossetti et al. 2009). Co-transfection of equal amounts of wild-type plasmids failed to completely 392 restore the 393 normal transcriptional activity. Since a reduced production of bioactive proteins was seen at western-blot, we 394 interpreted these results as consistent with a mechanism of haploinsufficiency similar to that described for the mutations 395 found in sheep. Among all the identified variations, only one was found to cause a premature truncation. This truncated 396 variant creates a premature stop in the pro-region (p.E211X), resulting in the complete lack of mature BMP15 peptide, 397 and was found in an Indian woman with PA and ovarian dysgenesis (Dixit et al. 2006). Very recently, a Chinese group 398 reported the first missense substitution (p.R329C) located in the region of the mature peptide that, involving an Arg to 399 Cys aminoacidic change, could alter the structure of BMP15 by impairing the correct folding of the protein (Wang et al. 400 2010). This variant co-segragated with POF phenotype in mother and daughter. To date, only two studies failed to find 401 an association between BMP15 mutations and POI: a Japanese group (Takebayashi et al. 2000) and a group from New 402 Zealand (Chand et al. 2006) which reported the absence of BMP15 mutations in series of women with SA. One possible 403 explanation for these negative results may be represented by the small size of the cohorts studied (15 and 38 POF 404 patients, respectively). Importantly, some of the missense variations in BMP15 gene have also been found in low 405 percentage in the control populations (see Table 2 for details), a finding that may question or diminish their pathogenic 406 role. In light of these findings, one could hypothesize that BMP15 variations might play a predisposing role in a context 407 of POI considered as a complex multifactorial disorder, in contrast with the view of POI as a monogenic disorder. 408 However, before drawing conclusions, it must be emphasized that the correct control population of these studies should 409 be represented by women of the same ethnicity and with proven physiological menopause beyond the age of 50 years. 410 Unfortunately, both these conditions were not met in most of the studies reporting BMP15 variants, as were instead in 411 the studies designed by our group (Di Pasquale et al. 2006; Rossetti et al. 2009). 412 The functional mechanism by which BMP15 variants with a proved biological impact may disturb ovarian 413 folliculogenesis is presently unknown. We may envisage that a diminished BMP15 paracrine signal in the follicle would 414 involve an impairment of the anti-apoptotic effects on GCs, a mechanism then favouring follicle atresia. Alternatively, 415 BMP15 variants may finally result in an altered recruitment of pre-antral follicles by gonadotrophins. For this reason, 416 BMP15 gene has also been investigated in patients with opposite alterations of ovulation mechanism, and no linkage 417 was found either in PCO patients and in mothers with spontaneous dizygotic twinning (Zhao et al. 2008). Indeed, 418 further studies are needed to understand the exact role of BMP15 variants in POI pathogenesis. Interestingly, all the 419 findings here described for several human variants might also suggest BMP15 as the first X-linked gene whose 420 haploinsufficiency may play a determinant role for the generation of ovarian dysgenesis in Turner syndrome, as 421 previously hypothesized by others (Layman 2006). Luca Persani, 2010

Androjenler folikül maturasyonunu çok erken fazlarda etkilerler Lenie ve Smitz androjenlerin alt nanomolar konsantrasyonlarda genomik ve non-genomik etkiler ortaya koyduğunu açıkladılar. Bir ligandla aktive olan transkripsiyon faktörü olarak, AR sitoplazmada sub-nanomolar androjenler bulur ve androjen sinyallerini gen ifadesinde değişikliklere dönüştürür . Androjenlerin hızlı olmayan genomik sinyallemesi sonuçta AR'ın transkripsiyonel aktivitelerini modüle edebilir. Ayrıca, çeşitli yumurtalık hücresi türleri arasında çoğu AR ifadesi granülosa hücrelerinde bulunabilir. Androjenler folikül maturasyonunu çok erken fazlarda etkilerler Androgen on ovary: Quite a paradox?? Role of androgen: Synergism with FSH Acts at early stages of maturation Instead of directly affecting the oocyte, it improves the environment. Get deeper into it later Let us look at the functions of the ovary once. Gleicher N, et al. Reproductive Biology and Endocrinology 2011, 9:116.

Figure 1 Mechanisms of androgen action and androgen biosynthesis. Mechanisms of androgen action and androgen biosynthesis. (A) Androgens can mediate their actions directly via the androgen receptor, or have indirect effects by their conversion into oestrogens and 3β-diol, which can activate the oestrogen receptor. DHT, dihydrotestosterone. (B) Androgen biosynthesis and metabolism. 3β-HSD, 3-β-hydroxysteroid dehydrogenase; 17β-HSD, 17β-hydroxysteroid dehydrogenase; 3β-diol, 5α-androstane-3β,17β-diol. K A Walters Reproduction 2015;149:R193-R218 © 2015 Society for Reproduction and Fertility

Ovaryum mikroçevresinin bozulması Azalan androgen seviyesi DHEA ‘ın folikülogenezde rolü yaş Ovaryum mikroçevresinin bozulması Azalan androgen seviyesi Zayıf oosit kalitesi IGF-1 Mitozun stimülasyonu granulosa ve theca hücrelerince steroid üretimi Normal oosit maturasyonu DHEA 2 major actions: steroidogenesis and follicular maturation. Now poor quality oocytes with age is basically because of decreasing oocyte environment and decreasing androgen levels. Supplementing the patient with androgen in the form of DHEA will improve the ovarian environment dramatically. This will act on whatever remaining androgen receptors are present. The action on the granulosa cells is interesting. It acts via its action increasing the level of Insulin like growth factor-1. This in turn stimulates mitosis– proliferation of the granulosa cells and also steroid production by the granulosa cells. This gives normal oocyte maturation and hormonal feedback to optimize the quantity of pituitary FSH. Eventually DHEA helps antagonize the adverse effects age has on the oocyte.

DHEA – etki mekanizması  AMH seviyesi  FSH reseptörleri Granulosa hücre apoptozunu önleme  Ovarian Steroidogenesis  Ovaryen yanıt  Oosit havuzunda artış  gebelik Sonmezer M, et al. Reprod Biomed Online. 2009; 19(4): 508-13.

TGFβ superfamily ; GDF9, BMP15, activin, inhibin, etc.

Figure 2 Ovarian cytokine and growth factor signalling factors – roles in primordial to primary follicle transition. Ovarian cytokine and growth factor signalling factors – roles in primordial to primary follicle transition. Green arrows are promoters of development and red arrows are inhibitors of follicle activation. Abbreviations: AMH, anti Müllerian hormone; BMP, bone morphogenic protein; FGF, fibroblast growth factor; GDF, growth differentiation factor; GDNF, glial-derived neurotrophic factor; KGF, keratinocyte growth factor; KL, KIT ligand; LIF, leukaemia inhibitory factor; SDF, stromal cell-derived factor. Eileen A McLaughlin, and Skye C McIver Reproduction 2009;137:1-11 © 2009 Society for Reproduction and Fertility

Figure 3 The oocyte KL – PTEN-PI3K – FOXO3 pathway governs follicle activation through control of initiation of oocyte growth. The oocyte KL – PTEN-PI3K – FOXO3 pathway governs follicle activation through control of initiation of oocyte growth. Abbreviations: KL, KIT ligand; KIT, CD117 (c-KIT); PIP2, phosphatidyl inositol bisphosphatel; PI3K, phosphoinositide 3-kinase; PIP3, phosphatidylinositol (3,4,5)-trisphosphate; PDK1-3, phosphoinositide-dependent protein kinase; Akt, protein kinase B; PTEN, phosphatase and tensin homolog; FOXO3, forkhead box O3A; GSK3, glycogen synthase kinase 3; CDKN1B, cyclin-dependent kinase inhibitor 1B (CDKN1); BMP15, bone morphogenic protein 15; Cx37, connexin 37; GDF9, growth differentiation factor 9. Eileen A McLaughlin, and Skye C McIver Reproduction 2009;137:1-11 © 2009 Society for Reproduction and Fertility

Reprodüktif hayatı etkileyen moleküler marker’ler (Liu et al,2005) Bir kadının üreme ömrü primordial folliküllerinin ömrü ile belirlenir. Foliküler rezerv, primer folikül havuzunda, fosfatidilinositol 3-kinaz (PI3K) ve fosfataz ve tensin homologunun (PTEN) salınması arasında karmaşık ve dikkatle kontrol edilen bir dengenin sağlanması yoluyla muhafaza edilir. Bu nedenle intra-oosit PI3K yolağının iki temel rolü vardır: folliküler aktivasyonda, aynı zamanda primordiyal foliküllerin sağkalımının sürdürülmesinde. PI3K/PTEN/Akt pathway is a key regulator of many normal cellular processes, including cell proliferation, survival, growth, motility, cytoskeletal rearrangement, and metabolism, through multiple downstream targets. Reprodüktif hayatı etkileyen moleküler marker’ler (Liu et al,2005)

Annu Makker1, Madhu Mati Goel1 and Abbas Ali Mahdi PI3K/PTEN/Akt and TSC/mTOR signaling pathways, ovarian dysfunction, and infertility: an update Annu Makker1, Madhu Mati Goel1 and Abbas Ali Mahdi Journal of Molecular Endocrinology doi: 10.1530/JME-14-0220 J Mol Endocrinol December 1, 2014 53 R103-R118

Figure 2 Schematic illustration of the principal genes known to be involved in POI pathogenesis and their site of expression in the ovary. Schematic illustration of the principal genes known to be involved in POI pathogenesis and their site of expression in the ovary. Luca Persani et al. J Mol Endocrinol 2010;45:257-279 © 2010 Society for Endocrinology

Figure 3 Hypothetical scheme of the dominant negative mechanism generated by the original BMP15 mutations described in the two heterozygous sisters affected with ovarian dysgenesis. Hypothetical scheme of the dominant negative mechanism generated by the original BMP15 mutations described in the two heterozygous sisters affected with ovarian dysgenesis. The recombinant p.Y235C variant was shown to produce aberrant products of high molecular weight by western blot performed in nonreducing conditions. These aberrant products secreted in the follicular fluid microenvironment may impair the paracrine signal of wild-type bioactive dimers by receptor sequestration, thus hampering the formation of dimers between types I and II BMP receptors and consequent generation of intracellular signal leading to the biological effects in target cells, i.e. granulosa cell growth and differentiation. Luca Persani et al. J Mol Endocrinol 2010;45:257-279 © 2010 Society for Endocrinology

Hsun-Ming Chang Jie Qiao Peter C.K. Leung Hum Reprod Update (2016) 23 (1): 1-18. Figure 2 Schematic diagram summarizing functional roles of BMPs and GDF9 in the human ovary. The potential physiological roles of intra-ovarian BMPs in regulating human ovarian functions, including steroidogenesis, activin production, cumulus–oophorus complex formation and expansion, cell–cell communication, ovulation and luteolysis are shown. BMP, bone morphogenetic protein; COC, cumulus–oophorus complex; Cx43, connexin 43; GC, granulosa cell; HAS2, hyaluronan synthase type 2; PTX3, pentraxin 3; StAR, steroidogenic acute regulatory protein. From: Oocyte–somatic cell interactions in the human ovary—novel role of bone morphogenetic proteins and growth differentiation factors Hum Reprod Update. 2016;23(1):1-18. doi:10.1093/humupd/dmw039 Hum Reprod Update | © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology.This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com 56

FSH-R gene mutations 6 different receptor mutations (566C----T) Aittomaki 1995, Beau 1998, Jiang 1998, Touraine 1999, Doherty2002 Finnish mutation Low E2 , High FSH and LH, Primordial and primer follicles in ovaries FSH-R gene mutations

Figure 4 Summary of putative intrafollicular roles of TGF-β superfamily members at different stages of follicle development. Summary of putative intrafollicular roles of TGF-β superfamily members at different stages of follicle development. Phil G Knight, and Claire Glister Reproduction 2006;132:191-206 © 2006 Society for Reproduction and Fertility

Over rezerv testleri(moleküler) Gonadotropinlerde nükleotid polimorfizmleri (SNP’ler) Bunların reseptör genleri,BMP-15,GDF 9, FMR1,MCM8 geni Over rezerv testleri(moleküler)

Tedavi seçenekleri > 40 yaş Comparison of ART options for women aged ≥ 40 Treatments compared Clinical pregnancy/cycle % Live birth/cycle % Study Clomiphene 0–4 % ~0 % Tsafrir et al. [25] Liu et al. [30] IUI with clomiphene 1–4 % <1 % 1–4.3 % Unreported Dovey et al. [29] IUI with FSH 3 % 1 % IVF 16.9 % 13.7 % Wiser et al. [39] Tedavi seçenekleri > 40 yaş

Melatonin and Ooycte Maturation Broad spectrum antioxidant, synthesized in pineal gland Most important antioxidant in ovarian follicle Does not promote oxidation under any circumstances Its metabolites are also capable anti-oxidants Enhances activity of other endogenous antioxidants like glutathione peroxidase & superoxide dismutase Synergy with other antioxidants such as Vitamins C & S Tamura H, et al. Journal of Ovarian Research. 2012; 5:5. ROS produced within follicles, especially during ovulation process, were scavenged by melatonin Reduced OS may be involved in oocyte maturation & embryo development Fernando S, et al. J Ovarian Res. 2014 Oct 21;7(1):98/ http://www.anti-aging.gr.jp/english/pdf/2012/9(1)0613.pdf

Melatonin ile ilgili kanıtlar ne söylüyor? Fernando S, et al. Journal of Ovarian Research. 2014; 7: 98.

Fernando S, et al. Journal of Ovarian Research. 2014; 7: 98.

Coenzyme Q10: Effects Coenzyme Q10 ATP production in mitochondria Restores normal energy production in ovary Antioxidant Protects from oxidative stress Bentov Y, et al. Fertil Steril. 2013; 99: 18-22/ Bentov Y, et al. Clinical Medicine Insights: Reproductive Heath. 2014; 8: 31-36.

DHEA - Adjunctive therapy in treatment of Infertility Prohormone Improves ovarian reserve Improves ovarian environment Augments IGF-1 Reduce apoptosis DHEA = Dihydroepiandrosterone Most abundant circulating steroid in humans Secreted by adrenal glands, testes & ovaries Synthesized from cholesterol Can be converted into other hormones- Estrogen & testosterone Levels of DHEA in women decline with age To summarize the actions of DHEA: DHEA represents the first compound in a new category of pharmacologic agents with potential to “rejuvenate” ovarian environments

Braz J Med Biol Res vol. 41 no. 11 Ribeirão Preto Nov Braz J Med Biol Res vol.41 no.11 Ribeirão Preto Nov. 2008  Epub Oct 31, 2008 http://dx.doi.org/10.1590/S0100-879X2008005000053  Braz J Med Biol Res, November 2008, Volume 41(11) 978-985 Effect of conditioned medium of mesenchymal stem cells on the in vitro maturation and subsequent development of mouse oocyte            B. Ling1,2, D.Q. Feng2, Y. Zhou2, T. Gao1, H.M. Wei3 and Z.G. Tian3 1Department of Obstetrics and Gynecology, 2Anhui Province Key Laboratory of Molecular Medicine, Anhui Provincial Hospital Affiliated to Anhui Medical University, Hefei, China  3Institute of Immunology, Hefei National Laboratory for Physical Science at Microscale and School of Life Science, University of Science and Technology of China, Hefei, China    

Mevcut literatür raporları ve meta-analiz sonuçları ROC eğrileri şu anda AMH ve AFC'nin prematür ovarian yetersizliğinde test olarak başarılı olduğunu göstermektedir Umut verici tahmini testler ve FSH bir Azalmanın değerlendirilmesi için yaygın olarak kullanılan tarama testi olarak görülmektedir Kritik seviyeye ulaşmadan önce yumurtalık rezervinde azalmanın saptanması önemlidir, önleyici antioksidan alımı ve sigaranın kesilmesi önemlidir Kötü over cevabını öngörmek için AFC, AMH ve bazal FSH testleri Moleküler testlerin henüz pratikte yer almadığı görülmekle birlikte önemi açıktır. FIGLA , FOXL2, BMP,LHR,FSHR mutasyonları,Telomeraz enzim ölçümleri,X kromozomunu ilgilendiren mutasyonlar Melatonin, CoQ, DHEA ve Vit C & E takviyeleri, kaybedilen yumurtalık fonksiyonlarını korumaya / tekrar kazanmaya yardımcı olur sonuçlar