AZF delesyonu olan infertil erkeklerde HIF-1α sinyal yolağının aktivitesinin araştırılması
Investigation of the activity of the HIF-1α signaling pathway in infertile men with AZF deletion
Yazar: DİLBESTE DEMİR
Danışman: DR. ÖĞR. ÜYESİ EMİNE NEDİME KORUCU
Necmettin Erbakan Üniversitesi / Fen Bilimleri Enstitüsü / Moleküler Biyoloji ve Genetik Ana Bilim Dalı
Derece: Yüksek Lisans, 2021
Konu: Biyoloji = Biology ; Genetik = Genetics ; Moleküler Tıp = Molecular Medicine
Dizin: Azospermi = Azoospermia ; Hipoksi = Hypoxia ; Hipoksi ile indüklenebilen faktör 1 alfa =   ; Mikrodelesyon = Microdeletion ; Y kromozomu = Y chromosome ; İnfertilite =

Özet:
İnfertilite üreme sisteminde tanımlanan bir hastalıktır. Dünya çapında %15 oranında görülmektedir. Erkeklerde infertilite temel olarak semen analizine göre belirlenir. Semen analizinde sperm hareketi, konsantrasyonu ve morfolojisi incelenerek WHO tarafından belirlenen referans değerlerine göre bireyin fertilite potansiyeli değerlendirilir. Fakat sadece semen analizi infertilitenin belirlenmesi için yeterli değildir. Gelişen teknoloji ile birlikte farklı analizler de yapılarak infertilite belirlenmektedir. Erkeklerde infertilite ürogenital enfeksiyon, konjenital, varikosel, immünolojik, cinsel faktörler ve endokrin bozukluklar gibi çeşitli nedenlerden kaynaklanmaktadır. Erkeklerde infertilitenin yaygın genetik nedenleri arasında Y kromozom mikrodelesyonları yer almaktadır. Y kromozomunda tanımlanan AZF bölgesinin delesyonu spermatogenez işlevini engelleyerek infertiliteye öncülük etmektedir. 
Hipoksi infertiliteye neden olan faktörler arasındadır. Hipoksi koşulları hücrelerde reaktif oksijen türlerini indüklemektedir. Reaktif oksijen türleri sperm hareketinden sorumlu olan ODF-1 proteini ile bağlantılıdır. Bu nedenle hipoksi erkek bireylerde sperm hareketini etkileyerek infertiliteye neden olmaktadır. Memeliler hipoksiye karşı yanıt oluşturarak hücre hasarından kurtulmaktadır. Hipoksiye karşı oluşturulan yanıt HIF-1 transkripsiyon faktörü ile gerçekleştirilmektedir. HIF-1 kompleksi hipoksik koşullar altında eritropoez, anjiogenez, apoptoz gibi çeşitli sinyal yolaklarını aktive eder. 
Çalışmamızda hipoksi ve erkek infertilitesi arasındaki ilişki araştırılmıştır. Elde edilen literatür bilgilerinden yola çıkarak erkek infertilitesinde hipoksiden sorumlu HIF-1α ile bağlantılı VEGFA, FLT-1, TNF-α ve ODF-1 düzeylerinin belirlenmesi amaçlanmıştır.
Sunulan araştırma Selçuk Üniversitesi Tıp Fakültesi Tıbbi Genetik Anabilim Dalı laboratuvarına gelen AZF delesyonuda olan 60 infertil erkek bireyden alınan kan örnekleri üzerinde gerçekleştirilmiştir. Kontrol grubu olarak sağlıklı ve 2-4 çocuk sahibi olan 30 erkek bireyden kan örnekleri alınmıştır. ODF-1 protein düzeyinin analizi sandviç Elisa yöntemiyle gerçekleştirilmiştir. HIF-1α ve bağlantılı genlerin ifadesi q-RT PCR analizi yoluyla yapılmıştır. 
Elde edilen sonuçlarda AZF delesyonuda olan infertil erkeklerde, sağlıklı kontrol grubuyla karşılaştırıldığında HIF-1α, TNF-α, VEGFA, FLT-1 genlerinin ekspresyon düzeyi yukarı regüle olduğu belirlenmiştir. ODF-1 protein düzeyinde kontrol grubuna göre anlamlılık (p˃0.05) görülmezken gen ekspresyonunda aşağı regülasyon görülmektedir. Çalışma sonucunda hipoksiden sorumlu olan HIF-1α'nın erkeklerdeki infertiliteden sorumlu olabileceği öngörülmektedir.

Abstract:
Infertility is a disease defined in the reproductive system. It is seen at a rate of 15% worldwide. Infertility in men is mainly determined by semen analysis. In the semen analysis, sperm movement, concentration and morphology are examined and the individual's fertility potential is evaluated according to the reference values determined by the WHO. However, semen analysis alone is not sufficient to determine infertility. With the developing technology, infertility is determined by making different analyzes. Infertility in men is caused by various causes such as urogenital infection, congenital, varicocele, immunological, sexual factors and endocrine disorders. Y chromosome microdeletions are among the common genetic causes of male infertility. Deletion of the AZF region defined on the Y chromosome leads to infertility by inhibiting the spermatogenesis function.
Hypoxia is among the factors that cause infertility. Hypoxia conditions induce reactive oxygen species in cells. Reactive oxygen species are associated with the ODF-1 protein, which is responsible for sperm motility. Therefore, hypoxia causes infertility by affecting sperm motility in male individuals. Mammals survive cell damage by responding to hypoxia. The response to hypoxia is mediated by the transcription factor HIF-1. The HIF-1 complex activates various signaling pathways such as erythropoiesis, angiogenesis, and apoptosis under hypoxic conditions.
In our study, the relationship between hypoxia and male infertility was investigated. Based on the literature information obtained, it was aimed to determine VEGFA, FLT-1, TNF-α and ODF-1 levels associated with HIF-1α responsible for hypoxia in male infertility.
The presented research was carried out on blood samples taken from 60 infertile male individuals with AZF deletion who came to the laboratory of the Department of Medical Genetics, Faculty of Medicine, Selcuk University. As the control group, blood samples were taken from 30 healthy male individuals with 2-4 children. Analysis of ODF-1 protein level was performed by sandwich Elisa method. Expression of HIF-1α and associated genes was determined by q-RT PCR analysis.
In the results obtained, it was determined that the expression level of HIF-1α, TNF-α, VEGFA, FLT-1 genes was upregulated in infertile men with AZF deletion compared to the healthy control group. ODF-1 protein level was not significant compared to the control group (p˃0.05), while gene expression was downregulated. As a result of the study, it is predicted that HIF-1α, which is responsible for hypoxia, may be responsible for infertility in men.

Infertility is a disease defined in the reproductive system. It is seen at a rate of 15% worldwide. Infertility in men is mainly determined by semen analysis. In the semen analysis, sperm movement, concentration and morphology are examined and the individual's fertility potential is evaluated according to the reference values determined by the WHO. However, semen analysis alone is not sufficient to determine infertility. With the developing technology, infertility is determined by making different analyzes. Infertility in men is caused by various causes such as urogenital infection, congenital, varicocele, immunological, sexual factors and endocrine disorders. Y chromosome microdeletions are among the common genetic causes of male infertility. Deletion of the AZF region defined on the Y chromosome leads to infertility by inhibiting the spermatogenesis function.
Hypoxia is among the factors that cause infertility. Hypoxia conditions induce reactive oxygen species in cells. Reactive oxygen species are associated with the ODF-1 protein, which is responsible for sperm motility. Therefore, hypoxia causes infertility by affecting sperm motility in male individuals. Mammals survive cell damage by responding to hypoxia. The response to hypoxia is mediated by the transcription factor HIF-1. The HIF-1 complex activates various signaling pathways such as erythropoiesis, angiogenesis, and apoptosis under hypoxic conditions.
In our study, the relationship between hypoxia and male infertility was investigated. Based on the literature information obtained, it was aimed to determine VEGFA, FLT-1, TNF-α and ODF-1 levels associated with HIF-1α responsible for hypoxia in male infertility.
The presented research was carried out on blood samples taken from 60 infertile male individuals with AZF deletion who came to the laboratory of the Department of Medical Genetics, Faculty of Medicine, Selcuk University. As the control group, blood samples were taken from 30 healthy male individuals with 2-4 children. Analysis of ODF-1 protein level was performed by sandwich Elisa method. Expression of HIF-1α and associated genes was determined by q-RT PCR analysis.
In the results obtained, it was determined that the expression level of HIF-1α, TNF-α, VEGFA, FLT-1 genes was upregulated in infertile men with AZF deletion compared to the healthy control group. ODF-1 protein level was not significant compared to the control group (p˃0.05), while gene expression was downregulated. As a result of the study, it is predicted that HIF-1α, which is responsible for hypoxia, may be responsible for infertility in men.