Human zona pellucida abnormalities

100 Pages·2016·1.89 MB·English

D OULU 2016
D 1379
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UNIVERSITY OF OULU P.O. Box 8000 FI-90014 UNIVERSITY OF OULU FINLAND
ACTA
A UNIVERSITATIS OULUENSIS
A C T A U N I V E R S I T A T I S O U L U E N S I S C
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A
D
MEDICA
Reeta-Maria Törmälä
Professor Esa Hohtola R HUMAN ZONA PELLUCIDA
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a ABNORMALITIES – A
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University Lecturer Santeri Palviainen
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r GENETIC APPROACH TO THE
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Postdoctoral research fellow Sanna Taskila ö UNDERSTANDING OF
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ä
lä FERTILIZATION FAILURE
Professor Olli Vuolteenaho
University Lecturer Veli-Matti Ulvinen
Director Sinikka Eskelinen
Professor Jari Juga
University Lecturer Anu Soikkeli
Professor Olli Vuolteenaho
UNIVERSITY OF OULU GRADUATE SCHOOL;
UNIVERSITY OF OULU,
FACULTY OF MEDICINE;
Publications Editor Kirsti Nurkkala
MEDICAL RESEARCH CENTER OULU;
OULU UNIVERSITY HOSPITAL;
ISBN 978-952-62-1297-5 (Paperback) NATIONAL GRADUATE SCHOOL OF CLINICAL INVESTIGATION
ISBN 978-952-62-1298-2 (PDF)
ISSN 0355-3221 (Print)
ISSN 1796-2234 (Online)

ACTA UNIVERSITATIS OULUENSIS
D Medica 1379
REETA-MARIA TÖRMÄLÄ
HUMAN ZONA PELLUCIDA
ABNORMALITIES – A GENETIC
APPROACH TO THE
UNDERSTANDING OF
FERTILIZATION FAILURE
Academic dissertation to be presented with the assent of
the Doctoral Training Committee of Health and
Biosciences of the University of Oulu for public defence in
Auditorium 4 of Oulu University Hospital, on 23
September 2016, at 12 noon
UNIVERSITY OF OULU, OULU 2016
Copyright © 2016
Acta Univ. Oul. D 1379, 2016
Supervised by
Professor Juha Tapanainen
Doctor Jouni Lakkakorpi
Reviewed by
Professor Sari Mäkelä
Docent Virpi Töhönen
Opponent
Professor Juha Kere
ISBN 978-952-62-1297-5 (Paperback)
ISBN 978-952-62-1298-2 (PDF)
ISSN 0355-3221 (Printed)
ISSN 1796-2234 (Online)
Cover Design
Raimo Ahonen
JUVENES PRINT
TAMPERE 2016
Törmälä, Reeta-Maria, Human zona pellucida abnormalities – a genetic approach
to the understanding of fertilization failure.
University of Oulu Graduate School; University of Oulu, Faculty of Medicine; Medical
Research Center Oulu; Oulu University Hospital; National Graduate School of Clinical
Investigation
Acta Univ. Oul. D 1379, 2016
University of Oulu, P.O. Box 8000, FI-90014 University of Oulu, Finland
Abstract
Despite the development of assisted reproduction technologies and significant advances in
reproductive biology and medicine over the years the cause of infertility remains unexplained in
10–20% of cases. The cause of infertility in these cases may be connected to problems in
fertilization or implantation and genetic factors may play a part in this.
The zona pellucida (ZP) is an extracellular matrix surrounding the oocyte and early-stage
embryos. It is important for folliculogenesis, fertilization and implantation. In humans, it is
composed of four known ZP glycoproteins that all show varying degrees of structural and
functional roles in reproduction. The aim of the present study was to examine the role of zona
pellucida genes in cases of total fertilization failure and zona anomalies, and to study their
expression in human fetal and adult ovaries.
A total of 34 sequence variations were detected in genes expressing the four human ZP proteins
(ZP1–ZP4) among women with fertilization failure and those with varying degrees of zona
anomalies in their oocytes. Most of the variations were known single nucleotide polymorphisms,
while three were novel findings. Women with fertilization failure had a higher mean number of
sequence variations in ZP1 and ZP3 when compared with controls. Some of the most frequent
zona anomalies may be at least partly explained by sequence variations in ZP1–ZP4 genes.
In fetal life, the expression of ZP3 protein and mRNA could already be detected as early as at
the 11th week of gestation and it peaked at the 20th week, the time of primordial follicle formation.
This suggests that components needed for zona matrix are already present well before the
formation of the zona pellucida and may have a role in the development of primordial follicles.
Expression of the transcription factor FIGLA (factor in the germline alpha) was increased at
around the 20th week of gestation, supporting previous findings of its critical role in the initiation
of folliculogenesis and primordial follicle formation.
The present study adds to our knowledge on the currently still incomplete picture of formation
of the ZP and fertilization in humans. Understanding the genetic background of infertile patients
may help us to develop new tools not only to evaluate but also to improve their fertilization
potential, and to choose the optimal treatment to achieve pregnancy.
Keywords: granulosa cell, infertility, oocyte, ovary, total fertilization failure, zona
anomaly, zona pellucida, zona pellucida glycoproteins 1–4

Törmälä, Reeta-Maria, Ihmisen alkiokuoren rakennehäiriöt – geneettiset tausta-
tekijät osasyynä hedelmättömyyteen?
Oulun yliopiston tutkijakoulu; Oulun yliopisto, Lääketieteellinen tiedekunta; Medical Research
Center Oulu; Oulun yliopistollinen sairaala; Valtakunnallinen kliininen tutkijakoulu
Acta Univ. Oul. D 1379, 2016
Oulun yliopisto, PL 8000, 90014 Oulun yliopisto
Tiivistelmä
Diagnostiikan kehityksestä huolimatta hedelmättömyyden syy jää edelleen epäselväksi 10–20
%:ssa tapauksista. Niissä hedelmättömyyden taustalla voivat olla munasolun hedelmöittymiseen
ja kohtuun kiinnittymiseen liittyvät ongelmat, jotka voivat osittain johtua geneettisistä syistä.
Alkiokuori on munasolua ja varhaista alkiota ympäröivä rakenne, joka osallistuu munarakku-
lan kehittymiseen, munasolun hedelmöittymiseen ja alkion tarttumiseen kohdun limakalvolle.
Ihmisellä alkiokuori muodostuu neljästä tunnetusta alkiokuoriproteiinista (ZP1–ZP4). Tutkimuk-
sessa selvitettiin alkiokuoriproteiineja koodittavien geenien vaikutusta hedelmällisyyteen poti-
lailla, joilla koeputkihedelmöitys ei ollut tuottanut yhtään hedelmöittynyttä munasolua (engl.
total fertilization failure, TFF) tai joiden munasoluissa havaittiin alkiokuoren rakennemuutoksia
(engl. zona anomalies, ZA). Lisäksi selvitettiin alkiokuoriproteiinien ja niiden lähetti-RNA:n
esiintymistä sikiöiden ja aikuisten munasarjoissa.
TFF- ja ZA-potilaiden alkiokuoriproteiineja koodittavista geeneistä löytyi yhteensä 34
nukleotidimuutosta. Muutoksista kolme oli uusia löydöksiä, mutta suurin osa oli ennalta tunnet-
tuja yhden nukleotidin polymorfioita eli geneettisiä monimuotoisuuskohtia. TFF-potilailla
havaittiin ZP1- ja ZP3-geeneissä keskimäärin enemmän polymorfioita kuin verrokeilla. Myös
osa yleisimmistä alkiokuoren rakennemuutoksista voidaan mahdollisesti selittää ZP1–ZP4-gee-
neistä löytyneillä polymorfioilla.
Sikiöllä ZP3:n ilmentyminen oli havaittavissa jo 11. raskausviikolla, mutta voimakkainta se
oli primordiaalivaiheen munarakkuloiden muodostumisen aikaan 20. raskausviikolla. Tämä voi
viitata siihen, että ZP3 saattaa osallistua primordiaalivaiheen munarakkulan kehittymiseen ennen
varsinaisen alkiokuoren muodostumista. ZP-geenien säätelytekijän FIGLA:n esiintyminen
lisääntyi 20. raskausviikolla, mikä tukee aikaisempia havaintoja FIGLA:n merkityksestä muna-
rakkulan kehittymisen aktivaatiossa ja primordiaalivaiheen munarakkuloiden muodostumisessa.
Tämä tutkimus tuo lisätietoa alkiokuoren merkityksestä munasolun hedelmöittymisessä ja
syventää tietämystämme alkiokuoren muodostumisesta ihmisellä. Hedelmättömyyden taustalla
olevien geneettisten tekijöiden tunteminen voi parantaa lapsettomuuspotilaiden hedelmällisyy-
den arviointia ja auttaa löytämään heille parhaiten sopivan hoidon.
Asiasanat: alkiokuoren rakennehäiriö, alkiokuori, alkiokuoriproteiini, granuloosasolu,
hedelmättömyys, munasarja, munasolu, zona pellucida glykoproteiini 1–4

Acknowledgements
This study was carried out at the Department of Obstetrics and Gynaecology,
University of Oulu and at the Clinical Research Center, Oulu University Hospital,
during the years 2003-2016.
First, I wish to express my deepest and sincere gratitude to my supervisor
professor Juha Tapanainen for his positive attitude, trust and endless
encouragement during this project. Juha has had a crucial role at every phase of my
thesis and I admire how he always found time for it despite his many commitments.
I am grateful for his patience during my long periods of parental leave and my
move abroad.
I wish to express my gratitude to my supervisor Jouni Lakkakorpi, Ph.D., for
his guidance throughout this project. I especially admire his writing skills, which
have given me a great advantage when writing articles and this thesis.
I am grateful to docent Tommi Vaskivuo for his excellent expertise with the
project that led to the second article. Professor Hannu Martikainen, Terhi Piltonen,
M.D., Ph.D. and docent Laure Morin-Papunen are acknowledged for their
encouragement and valuable comments during our research meetings.
I wish to thank docent Minna Männikkö, docent Timo Tuuri, Anni Haltia Ph.D.,
professor Leena Ala-Kokko, Annikki Liakka, M.D., Ph.D. and Sinikka Nuojua-
Huttunen M.D., Ph.D., for their valuable collaboration.
I warmly thank professor Markku Heikinheimo for kindly inviting me to his
innovative research group for one year. Helka Parviainen and the rest of the
research group are acknowledged for their support and great company during and
after that year.
I have had the privilege and joy to work on my thesis in an inspiring and warm-
hearted research group. I am grateful to Minna Jääskeläinen, Johanna Puurunen,
Sanna Koskela, Kristiina Mäkelä, Mervi Haapsamo, Zdravka Veleva and Outi
Uimari for their support and friendship in and out of the lab.
I wish to thank professor Sari Mäkelä and docent Virpi Töhönen for their
careful review of this thesis. I thank Nicholas Bolton for his excellent linguistic
revision of this thesis and the manuscripts over the years. I warmly thank Mirja
Ahvensalmi for her skilful assistance in the laboratory. Seija Leskelä is
acknowledged for her talented figure editing and her friendly attitude. I also want
to thank Risto Bloigu for helping with the statistics.
I want to thank my very dear friends and research fellows Anna Hakalahti,
Päivi Honkavaara, Irina Nagy and Päivi Fonsén for being there for the ups and
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downs of the research work and for all the great moments we have had together. I
wish to thank Ingrid Huzen for her friendship and encouragement. I want to thank
my soul mate Laura Myllymäki for her love and support over the years.
I warmly thank my parents Anneli and Pertti for their support and endless
willingness to help. I am very grateful to my father who started regularly
babysitting Kerttu (and later also Hannes) when she was nine months old so that I
could go on with my work. I wish to thank my sister and dear friend Kaisa for her
encouragement in work and in life. My brother Juho and his partner Sanna are
thanked for the nice moments together. I thank my sister-in-law Nina and her
husband Petri for their friendship.
I want to thank Panu for his endless love, support and help during my work on
this thesis. I am deeply grateful for the adventures in life that we have taken together.
I thank Kerttu and Hannes, the treasures of my life, for teaching me how the beauty
of life lies in the small things and how to live in the moment.
This study was financially supported by the Academy of Finland, the Sigrid
Jusélius Foundation, the Finnish Cultural Foundation, the Foundation of the
University of Oulu and Oulu University Hospital, who are gratefully acknowledged.
Amstelveen, June 2016 Reeta Törmälä

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