ACTAUNIVERSITATIS

UPSALIENSISUPPSALA

2014

Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Medicine 1048

Biological Markers of Fertility

SARAH NORDQVIST

ISSN 1651-6206ISBN 978-91-554-9085-0urn:nbn:se:uu:diva-234067

Dissertation presented at Uppsala University to be publicly examined in Sal IX,Universitetshuset, Övre Slottsgatan 2, Uppsala, Friday, 5 December 2014 at 09:15 for thedegree of Doctor of Philosophy (Faculty of Medicine). The examination will be conductedin English. Faculty examiner: Professor Geraldine Hartshorne (University of Warwick).

AbstractNordqvist, S. 2014. Biological Markers of Fertility. Digital Comprehensive Summaries ofUppsala Dissertations from the Faculty of Medicine 1048. 74 pp. Uppsala: Acta UniversitatisUpsaliensis. ISBN 978-91-554-9085-0.

Infertility affects 15 % of couples, which corresponds to 60 - 80 million worldwide. Themicroenvironments in which the oocyte, embryo and fetus mature are vital to the establishmentand development of a healthy pregnancy. Different biological systems, such as angiogenesis,the immune system and apoptosis need to be adequately regulated for pregnancy to occur andprogress normally. The overall aim of this thesis was to investigate the impact of Histidine-rich glycoprotein (HRG) and Src homology 2 domain-containing adapter protein B (SHB) onhuman female fertility.

HRG is a plasma protein that regulates angiogenesis, the immune system, coagulation/fibrinolysis and apoptosis, by building complexes with various ligands. The impact of HRGon fertility is studied here for the first time. HRG is present in follicular fluid, the Fallopiantube, endometrium, myometrium and placenta. HRG distribution within embryo nuclei dependson developmental stage. Blastocysts express and secrete HRG. The HRG C633T singlenucleotide polymorphism (SNP) appears to affect the chance of pregnancy and, correspondingly,parameters associated with pregnancy in IVF. Additionally, this HRG genotype may increasethe risk in IVF of only developing embryos unfit for transfer.

SHB is an adaptor protein involved in intracellular signaling complexes that regulateangiogenesis, the immune system and cell proliferation/apoptosis. Shb knockout mice havealtered oocyte/follicle maturation and impaired embryogenesis. The impact of three SHBpolymorphisms (rs2025439, rs13298451 and rs7873102) on human fertility is studied for thefirst time. The SNP prevalences did not differ between infertile and fertile women. BMI,gonadotropin dosages, the percentage of immature oocytes, the number of fertilized oocytes,the percentage of good-quality embryos and the day of embryo transfer seems to be affectedby SHB genotype.

In conclusion, HRG and SHB appear to influence female fertility. They are potentialbiomarkers that might be used for predicting pregnancy chance in infertile women. Knowledgeof these genotypes may improve patient counseling and individualization of treatment.

Keywords: angiogenesis; embryogenesis; female reproductive tract; fertility; fertilization;Histidine-rich glycoprotein; intron; in vitro fertilization; oocyte; ovarian response; pregnancy;single nucleotide polymorphism; Src homology 2 domain-containing adapter protein B

Sarah Nordqvist, Department of Women's and Children's Health, Akademiska sjukhuset,Uppsala University, SE-75185 Uppsala, Sweden.

© Sarah Nordqvist 2014

ISSN 1651-6206ISBN 978-91-554-9085-0urn:nbn:se:uu:diva-234067 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-234067)

List of Papers

This thesis is based on the following papers, which are referred to in the text by their Roman numerals.

I Nordqvist, S., Kårehed, K., Hambiliki, F., Wånggren, K.,

Stavreus-Evers, A., Åkerud, H. (2010) The presence of histi-dine-rich glycoprotein in the female reproductive tract and in embryos. Reprod Sci, 17:941-947.

II Nordqvist, S., Kårehed, K., Stavreus-Evers, A., Åkerud, H. (2011) Histidine-rich glycoprotein polymorphism and pregnan-cy outcome: a pilot study. Reprod Biomed Online, 23:213-219.

III Nordqvist, S., Kårehed, K., Skoog Svanberg, A., Menezes, J., Åkerud, H. (2014) Ovarian response is affected by a specific Histidine-rich glycoprotein polymorphism – a preliminary study. Reprod Biomed Online, in press (Acceptance letter Sept. 24, 2014).

IV Nordqvist, S., Åkerud, H., Kårehed, K. (2014) The effect of SHB gene polymorphisms on ovarian response and oocyte mat-uration in IVF. Submitted.

Reprints were made with permission from the respective publishers.

Contents

Introduction ..................................................................................................... 9 Historical perspective on fertility ............................................................... 9 Current knowledge of fertility .................................................................. 10 

Pathophysiological aspects of female fertility ..................................... 10 Causes of Infertility ............................................................................. 13 Clinical diagnostics .............................................................................. 14 IVF ....................................................................................................... 15 Genetic and biochemical markers of fertility ...................................... 17 

Aims .............................................................................................................. 27 

Material and methods .................................................................................... 28 Paper I and II ............................................................................................ 28 

Study participants ................................................................................ 28 Methods ............................................................................................... 29 

Paper III and IV ........................................................................................ 30 Study participants ................................................................................ 30 Methods ............................................................................................... 30 

Nomenclature of HRG polymorphism ..................................................... 31 Statistical analysis .................................................................................... 31 Ethical Approval ...................................................................................... 31 

Results ........................................................................................................... 32 Paper I ...................................................................................................... 32 

HRG in follicular fluid ......................................................................... 32 HRG in culture medium and embryos ................................................. 32 HRG in the female reproductive tract .................................................. 32 

Paper II ..................................................................................................... 33 Pregnancy results related to HRG protein isoform or genotype .......... 33 

Paper III .................................................................................................... 34 Demographic variables ........................................................................ 34 Treatment background/parameters ...................................................... 34 Laboratory parameters ......................................................................... 35 

Paper IV ................................................................................................... 35 Allele frequencies, haplotypes and linkage disequilibrium ................. 35 Demographic variables ........................................................................ 36 Treatment parameters .......................................................................... 37 

Laboratory parameters ......................................................................... 37 

Discussion ..................................................................................................... 41 Methodological considerations................................................................. 41 HRG and female fertility .......................................................................... 42 

Ovaries and oocytes ............................................................................. 42 Follicular fluid ..................................................................................... 43 Fertilization .......................................................................................... 44 Embryo development and implantation/placentation .......................... 44 Proposed mechanisms of HRG function in female reproduction ........ 45 

SHB and female fertility .......................................................................... 46 Ovaries and oocytes ............................................................................. 47 Embryo development ........................................................................... 49 

Conclusions ................................................................................................... 50 

Clinical Implications, at present and in the future ........................................ 51 

Summary in Swedish – Sammanfattning på svenska ................................... 53 

Acknowledgements ....................................................................................... 55 

References ..................................................................................................... 57 

Abbreviations

AMH Anti-Müllerian hormone AFC Antral follicle count bFSH Basal FSH BMI Body mass index C/C Homozygous for the HRG C633 SNP C/T Heterozygous for the HRG C633T SNP ECM Extracellular matrix ERK Extracellular regulated kinase ET Embryo transfer FAK Focal adhesion kinase FGF Fibroblast growth factor FSH Follicle-stimulating hormone GV Immature oocyte, germinal vesicle hCG Human chorionic gonadotropin HS Heparan sulfate hMG Human menopausal gonadotropin HRG Histidine-rich glycoprotein ICSI Intracytoplasmic sperm injection IGF Insulin growth factor IVF In vitro fertilization LD Linkage disequilibrium LH Luteinizing hormone MI Immature oocyte, metaphase I MII Mature oocyte OPU Ovum pick-up PTB Phosphotyrosine binding domain rFSH Recombinant FSH SHB Src homology 2 domain-containing adapter

protein B SH2 Src homology 2 region SNP Single nucleotide polymorphism TSP Thrombospondin T/T Homozygous for the HRG 633T SNP VEGF Vascular endothelial growth factor

9

Introduction

Historical perspective on fertility Female fertility has intrigued mankind throughout time. Humans have sought to cure infertility through a rich variety of treatments. There are Egyptian documents from 1900 BC discussing the diagnosis and treatment of gynecological problems. However, it was not until the Renaissance that a scientific approach began to go beyond magical or religious explanations. DaVinci (Corporis Fabrica, 1543) and others began to study female anato-my during this period. In 1677, sperm was discovered by van Leeuwenhock. It took more than a century for van Baer to discover the oocyte (1827, ‘De ovi mammalium et homonis genesi’) (1).

The late 19th and early 20th century brought further insights when the pro-cess of fertilization was described by Nelson in 1852. The connection be-tween menstrual disorders and fertility was also revealed. In the 1920s hor-mones, such as estrogen and progesterone, were discovered (2) and physi-cians finally recognized the association between poor sperm counts and in-fertility.

The era of in vitro fertilization (IVF) began when Pincus conducted IVF experiments with rabbits in 1934 (3). Rock claimed to successfully fertilize human oocytes in vitro 10 years later (4), though it may have been partheno-genic activity (5). Gemzell developed the usage of gonadotropins for ovula-tion induction in the late 1950’s and early 1960’s (6). In 1975, Edwards and Steptoe (7) achieved the first verified IVF pregnancy but it resulted in an ectopic pregnancy. The first baby born through IVF was in 1978 (8). There-after, this technique spread throughout the world, bringing further advances. Though the first mouse from a cryopreserved embryo came in 1972, the first human birth was in 1984 (9). Pre-implantation genetic screening (PGS) was developed in 1989 (10). In the early 1990s, intracytoplasmic sperm injection (ICSI) was discovered (11). A proud year for all involved in IVF occurred 2010, when Robert Edwards was awarded the Nobel Prize in medicine. It is estimated that there are now five million children conceived by IVF world-wide (12).

10

Current knowledge of fertility Dorland’s Medical Dictionary (13) defines fertility as “the capacity to con-ceive or induce conception”. The chance of pregnancy the first month for a couple having unprotected intercourse at ovulation is about 20 % (14). Time to conception, the fertility rate, can be used to measure fertility. Sixty per-cent of couples will conceive within six months, 80 – 90 % within a year and 90 – 95 % within two years (15). Infertility is clinically defined as the failure to achieve a pregnancy after at least one year of regular unprotected sexual intercourse (16). Approximately 15 % of couples of a fertile age suf-fer from infertility, which corresponds to 60 - 80 million couples worldwide (17).

Pathophysiological aspects of female fertility

The ovaries and oocyte maturation A woman undergoes approximately 400 - 500 ovulations during her lifetime. However, she will produce 6 - 7 million oogonia. At birth, there are 500 000 - 2 million oocytes; at puberty, 300 000 - 500 000; and at menopause, 100 - 1000. Thus, there is an immense loss of oogonia and oocytes, beginning in the early fetus and continuing until menopause.

Oocytes develop from primordial germ cells that have migrated around the fourth embryonic week from the yolk sack to the gonadal ridge. The exact cell of origin and the factors initiating, sustaining or steering migration are unknown. However, molecules within the local microenvironment, such as kit-ligand, transforming growth factor-β1, and laminin are involved (18). The germ cells undergo mitosis repeatedly. Reaching the gonadal ridge, they become surrounded by supporting epithelial and mesenchymal cells and form colonies.

Germ cells differentiate into oogonia, reaching a maximum at 16 - 20 weeks and thereafter decline rapidly. Several known and unknown processes are needed for oogonia to enter the first meiosis and become oocytes. Genes, such as WNT4 and RSPO1, are important as well as retinoic acid for this development (19). Upon entering the first meiotic division, oocytes become arrested in the diplotene stage of prophase.

Vascular channels and perivascular cells begin to appear in the ovarian cortex at 18 - 20 weeks. Perivascular cells then become pre-granulosa cells that surround the oocytes in a single layer, forming primordial follicles. The signal stimulating continued development of primordial follicles is not fully elucidated, though it is independent of follicle-stimulating hormone (FSH) (18). Activation of primordial follicle development is suppressed by anti-Müllerian hormone (AMH), inhibin, tensin-homolog deleted on chromosome 10 (PTEN) and is activated by the mammalian target of rapamycin

11

(mTORC) (20). The pool of primordial follicles may also regulate recruit-ment, perhaps through paracrine regulation (20) or even the spatial arrange-ment of follicles (21).

When the pre-granulosa cells become cuboidal, a primary follicle is formed. Next, granulosa cells form several layers around the oocyte and mesenchymal cells differentiate into a loosely packed theca cell layer, devel-oping into pre-antral follicles. The layer of theca cells becomes more defined and contains blood vessels. A fluid-filled space forms and the follicle be-comes an antral follicle. Primary follicles take approximately 85 days to mature. From the antral follicle stage forward, this process is dependent on gonadotropins. Granulosa cell proliferation is increased around the oocyte, protruding into the increasingly fluid-filled space, forming the cumulus oo-phorous. The theca cell layer matures and becomes more vascularized.

The communication between granulosa cells and the oocyte is important for oocyte development. It occurs through gap junction and paracrine signal-ing (22). These processes allow for exchange of oxygen, nutrients, metabo-lites and necessary signaling molecules. Oocyte maturation will cease if this communication is interrupted (23, 24).

Nearing ovulation, the theca layer will become avascular. The mid-cycle surge of luteinizing hormone (LH) increases prostaglandins and proteases, such as plasmin. The proteolytic enzymes degrade the follicle wall, allowing for the expulsion of the cumulus oophorous. Prostaglandins activate the epi-dermal growth factor-like signaling pathway (25). This pathway is involved in the expansion of the cumulus cells and initiates the resumption of meiosis. The oocyte completes the first division of meiosis and enters the second meiosis division, arresting at the second metaphase. Prior to ovulation, the gap junctions between granulosa cells and the oocyte become disrupted and the cumulus oophorous completely detaches from the follicle wall.

After ovulation, the follicle becomes converted to a corpus luteum cyst, responsible for the production of progesterone. The granulosa cells enlarge, filling the cystic cavity and undergo luteinization. The corpus luteum also becomes vascularized.

The Fallopian tubes and oocyte fertilization After ovulation, the oocyte/cumulus complex adheres to the ovary until the Fallopian tube fimbria moves over the oocyte. Muscular contractions and movement of the cilia sweep the oocyte into the Fallopian tube. Molecules from follicular fluid, released at ovulation, may affect cilia movement and transportation of the oocyte (26). The Fallopian tube also contains non-cilia epithelium, which secretes different factors throughout the menstrual cycle. Growth factors or cytokines, seem important for transportation and fertiliza-tion of the oocyte and, also, for transportation, development and implanta-tion of the embryo when it enters the uterus (27).

12

The oocyte is fertilized at the isthmus-ampulla junction about 12 - 24 hours after ovulation. Only several hundred sperm will reach this location. An unknown chemical substance attracts sperm to the oocyte (28). Factors secreted by luminal epithelium may be important for the competency of the sperm (29, 30). Normally, only one sperm will fertilize the oocyte. Upon fertilization, the oocyte completes the final stages of meiosis. Early cleavage is primarily dependent upon mRNA and proteins inside the oocyte. At the 4 - 8 cell stage, the embryos will start to express its own genes (31) and produce substances needed for development. The embryo will remain in the Fallopian tube until the morula stage.

The uterus and implantation After entering the uterine cavity, the morula will develop into a blastocyst, hatch and shed its zona pellucida. In vitro, this is achieved by the blastocyst expanding and contracting. However, in vivo, hatching is probably caused by lyses from substances contained within the uterine fluid or excreted from the blastocyst (32).

The embryo and the endometrium have an intricate cross-communication. There is a vast array of signaling molecules involved in this communication, which is essential for successful implantation. The embryo is believed to produce a factor that stimulates the ovaries to produce early pregnancy factor (EPF) within 1 – 2 days after fertilization. Additionally, the embryo produc-es human chorionic gonadotropin (hCG) before implantation. The endome-trium has a window of receptivity around day 7 - 9 after ovulation. During this period, pinopods appear on the endometrium surface and remove fluid in the uterine cavity, thus improving contact with the blastocyst. They also remove mucin, an anti-adhesion molecule. Several adhesion molecules, such as integrins, selectins and trophinin, are involved in implantation. Following adhesion, metalloproteinases degrade the extracellular matrix (ECM), allow-ing trophoblasts to invade. There is a local increase in cytokine-producing lymphocytes and macrophages. Cytokines such as CSF-1, LIF and IL-1 are necessary for implantation (33). Also, hormones, growth factors and angio-genic factors are important substances in this process (34).

13

Causes of Infertility Causes and frequencies of infertility are shown in Figure 1.

Figure 1. Causes of infertility (35).

Ovulation disturbances About 17 % of infertility is caused by ovulation disturbances (35). Polycys-tic ovary syndrome is the most common cause, with a prevalence of 87 % among women with oligomenorrhea (36). Excessive exercise, stress or weight loss/gain, thyroid disease, hyperprolactinemia, Cushing’s, Addison’s disease, brain lesions and congenital adrenal hyperplasia can also cause hormonal imbalances leading to oligomenorrhea.

Tubal factors Tubal factors account for about 14 % of all female infertility (37). Women with previous pelvic inflammatory disease, endometritis, endometriosis, ectopic pregnancy, ruptured appendix, previous abdominal surgery or in-flammatory bowel disease have an increased risk of tubal impairment. Other causes of dysfunction can be due to other abnormalities such as tubal dys-genesis or cystic fibrosis. Damage may be due to complete or partial obstruc-tions or to poor motility. This prevents oocyte capture at ovulation or oo-cyte/embryo transportation.

Uterine factors There is an increased prevalence of submucosa uterine fibroids; intrauterine synechiae, caused by previous surgery or infections; and endometrial polyps in infertile women (38).

Endometriosis Endometriosis is when endometrial epithelial or stromal cells develop out-side the uterus. This can cause ovarian cysts, peritoneal bleeding and infertil-ity by affecting ovarian, tubal and/or uterine function. The pathogenesis is unclear. However, it is an inflammatory condition with alterations in im-

14

mune function with cytokine production contributing. Additionally, there may be impaired apoptosis (39).

Endometriosis accounts for 15 % of female infertility and its prevalence is 10 % within the general population. Monthly fecundity is 2 – 10 % in wom-en with endometriosis. Spontaneous conception within three years are lower in women with endometriosis (36 %) compared with those with unexplained infertility (54 %) (40).

Male factor Approximately 26 % of infertility is due to a male factor. The World Health Organization has recommended reference levels for sperm tests, though these are controversial (41). Age, smoking, obesity, medications, recreation-al drugs, toxic substances, surgery, trauma, infections, systemic diseases and genetic abnormalities can reduce male fertility either by affecting sperm quality and/or male potency (42). Sexual dysfunction is the primary cause of infertility for five percent of couples seeking treatment (43).

Unexplained infertility The diagnosis “unexplained” is applied when the screening has not found any cause of infertility. It is dependent upon the tests performed and the in-terpretation of the findings. Thus, this diagnostic group may have couples with undiscovered causes such as endometriosis, premature ovarian aging, etc. (44). Additionally, couples with unexplained infertility may represent those with a normal fertility who are at the lower end of a normal distribu-tion because many will conceive spontaneously (45). However, 18.5 % will not achieve an ongoing pregnancy even after undergoing infertility treatment (45). Thus, unexplained infertility may represent a population for which current diagnostic tools are inadequate. Unknown angiogenic, immunologi-cal, genetic or endometrial defects could be the cause, affecting gametogene-sis, fertilization, implantation or pregnancy.

Also noteworthy is the large number of patients with multiple causes of infertility (13.9 %) (35). Unknown defects might also exist in couples with an infertility diagnosis.

Clinical diagnostics Diagnostic tests Ovulation can be tested in several ways. Regular menstrual cycles indicate probable ovulation. The LH surge prior to ovulation can be indicated in urine samples. Vaginal ultrasound can detect a dominant follicle. One week after ovulation, progesterone levels reach a peak, indicating a well-functioning corpus luteum and that ovulation has occurred. The most common blood

15

tests for anovulation are thyroid tests and prolactin, as well as basal FSH (bFSH) and LH taken cycle day 3 - 5 (46).

Ovarian reserve is the amount of remaining oocytes within the ovaries (20). Aging decreases the number of oocytes. Fertility begins declining ap-proximately 20 years before menopause and a loss of fertility occurs about 10 years before menopause (47). A low initial reserve or an accelerated de-pletion will shorten the number of fertile years (48). Ovarian reserve testing can be accomplished by several methods (49) including bFSH and AMH levels, and ultrasound measurements of ovarian volume and/or the number of antral follicles (AFC). AFC and AMH are emerging as the best tests of ovarian reserve (50, 51). However, none of these measurements are precise (49) and it is debated whether or not they can predict the possibility of live birth.

Oocyte quality is difficult to assess, even in an IVF laboratory. Certainly, all healthy children originate from a good-quality oocyte. Indirect measures that predict chance of pregnancy probably also, predict the chance of obtain-ing a good-quality oocyte. Morphological assessment of oocytes has been attempted but is controversial because most studies are small and results are conflicting (52).

Tubal factors can be suspected based on medical history and tools for in-vestigation include laparoscopy, hysterosalpingography and hysterosalpin-gosonography. Uterine causes of infertility can be discovered by vaginal ultrasound, hysterosalpingography, hysterosonography or hysteroscopy.

IVF Controlled ovarian hyper-stimulation induces the ovaries to produce several mature oocytes. Typically, this is achieved by daily gonadotropin injections for approximately two weeks. Dosages are often prescribed individually based on anticipated ovarian response. Ultrasound monitoring usually begins stimulation day 6 – 10 and is repeated as necessary until a certain number of follicles reach a specific size.

There are two methods to prevent spontaneous ovulation: Gonadotropin releasing hormone (GnRH) agonist. This is often giv-

en as a spray begun two weeks prior to gonadotropin initiation and usually continued until ovulation induction.

GnRH antagonists. The gonadotropin injections are begun in the be-ginning of menstruation. The antagonist, given as daily injections, is begun either on a fixed day, usually about day 5 - 6, or when the leading follicle reaches a size of 14 mm.

When the leading follicles reach a mature size, ovulation is induced, commonly with an hCG injection, which mimics the LH trigger. Ovum pick-up (OPU) is timed 35 - 38 hours after the hCG injection. The oocytes and follicle fluid are aspirated from each follicle using a needle attached to the

16

vaginal ultrasound probe. The oocytes are placed in culture medium and transferred to an incubator.

There are two different techniques for fertilization of the oocyte. In stand-ard IVF, prepared sperm in a fixed concentration are added to the culture medium containing oocytes. ICSI is often used with low sperm counts or previously low fertilization rates. For ICSI, the cumulus cells are removed from the oocyte and maturity is assessed before a single sperm is injected into each oocyte.

The day after OPU, fertilization is assessed. Additionally, oocyte maturity is determined in standard IVF oocytes. Embryos are cultured 2 - 6 days. Embryos are assessed in accordance with standard clinical morphological scoring protocol (53) and a good-quality embryo is chosen for embryo trans-fer (ET). The remaining good-quality embryos are cryopreserved. Approxi-mately 74 % of fresh embryo transfers are single ET and 26 % double ET in Sweden (2012, http://www.ucr.uu.se/qivf/index.php/ behandlingsresultat, 140721). Luteal phase support is given by vaginal progesterone administra-tion. Pregnancy testing is done 14 - 21 days after ET.

IVF has a higher chance for pregnancy compared with treatment alterna-tives such as ovulation stimulation or insemination, which have pregnancy rates between 5 - 20 % (54). Clinical pregnancy results after IVF in Sweden are presented in Figure 2. Nationally, standard IVF and ICSI usage is about equal. Approximately 87 % of fresh embryo transfers are performed on day 2 - 3 (www.ucr.uu.se/qivf/index.php/behandlings-resultat, 20140721). More than 60 % of couples in Sweden have a child after undergoing up to three IVF treatments (55).

Figure 2. Clinical pregnancy rate per IVF treatment with fresh embryo transfer in Sweden 2012. Adapted from www.ucr.uu.se/qivf/index.php/behandlings-resultat, 20140721.

Factors that predict the chance of pregnancy during IVF/ICSI Models using prognostic variables have been developed to predict pregnancy in IVF (56), although they are not always universally applicable because clinical routines and laboratory practices differ (56). Demographic variables (for example, age, infertility duration, smoking, number of previous preg-

17

nancies or births, menstrual cycle length and body mass index (BMI)), clini-cal/diagnostic variables (for example, bFSH, AMH, AFC, infertility diagno-sis, number of previously unsuccessful attempts and gonadotropin dosage) and also laboratory parameters (for example, fertilization method, number of oocytes, embryo quality, day of transfer, number of embryos transferred and number of good-quality embryos) have been associated with pregnancy out-come after IVF (56-63).

Laws, regulations and accessibility Healthcare structure, regulations/laws and costs affect access to fertility treatment and indirectly an individual’s chance of conceiving (64). Com-pared with other countries, access to reproductive treatment in Sweden is good (46). IVF is publicly funded for most couples without children. Cur-rently, the number of treatments allowed and eligibility criteria for publicly financed treatment vary between regional healthcare authorities. Couples are required to live in a stable relationship (Law 2006:351, Chap. 7 3§). Also, double embryo transfer is allowed only if the risk for twins is deemed mini-mal. Transfer of more than two embryos is never allowed (SOSFS 2005:17). Embryo donation, surrogacy or treatment of single adults is not yet allowed (46).

Genetic and biochemical markers of fertility The whole human genome was sequenced between 1990 - 2003 (http://web. ornl.gov/sci/techresources/Human_Genome/project/timeline.shtml, 140824), just 50 years after Watson and Crick’s article on DNA was published (65). Computer technology advancements have enabled complex analysis within fields such as bioinformatics and statistics. This is revolutionizing our under-standing of genetics and microbiology. Internet accessibility has also facili-tated the sharing of information about genes through public databases.

Many current studies are examining variations in genomics, transcriptom-ics, proteomics, secretomics and metabolomics to discover more about re-productive pathophysiology, diseases, diagnostic tools and innovative new treatments. One expanding field within genomics is the study of single nu-cleotide polymorphisms (SNP). These are common genetic variations within a population.

Genetic variations associated with infertility Polymorphisms of genes have been associated with infertility. IVF gonado-tropin requirements (66), poor or high responders (67, 68), the number of mature oocytes, fertilization rates, good-quality embryos and pregnancy (69) have been linked to various polymorphisms such as the FSH receptor (69), estrogen receptor (70-72) and folate-metabolizing pathway genes (73). Pro-gesterone levels and pregnancy differences have been connected with the

18

Scavenger receptor class B type 1 gene polymorphisms (74). SNPs in angio-genic factors, such as vascular endothelial growth factor (VEGF), are associ-ated with recurrent IVF failure (75). Even genetic variations, such as in the hCG Beta gene (76), appear to prevent miscarriage. Thus, it has been sug-gested that individualizing treatment to patients’ genotypes could improve outcome (77).

Histidine-rich glycoprotein (HRG) Histidine-rich glycoprotein (HRG) is an abundant multi-domain plasma gly-coprotein produced mainly in liver parenchymal cells (78). HRG is trans-ported both as a free protein in serum and in platelet granules (79). The pro-tein is present in plasma in relatively high concentrations (100 - 150 µg/ml) (80). Newborns have 20 % of adult levels and levels increase with age (81). Families with congenital HRG deficiencies have approximately 20 - 35 % of normal plasma HRG level. Members of these families have no abnormal hemostatic or immunological function so it has been proposed that 20 % of normal levels are enough for adequate physiological functioning (82). HRG may be a negative acute phase reactant because HRG levels decrease with increasing CRP levels (83).

HRG regulates tumor growth (84-86). HRG plasma levels in breast cancer patients are elevated (87). In ovarian cancer patients, the levels decrease with increasing stage (88). HRG protein expression is down-regulated in serum from endometrial hyperplasia and carcinoma patients (89).

Molecular structure HRG consists of a 525 amino acid polypeptide (sometimes described as 507 if the 18 amino acid signal peptide is not included) with three main domains as shown in Figure 3 (90). Two cystatin-like regions (N1 and N2) are posi-tioned at the N-terminal. These domains are followed by a histidine-rich region (HRR), flanked by two proline-rich regions (PRR) and a C-terminal domain (80). The protein contains at least six disulphide bonds (80, 91). HRG has several glycosylation sites (80, 90, 92) and a recent study suggests that HRG might be phosphorylated (93). HRG is usually negatively charged in normal physiological conditions, but becomes positively charged in mild-ly acidic conditions or through binding to Zn2+ (80). If positively charged, this causes a conformational change in molecular structure through a change in the disulphide bond (Cys 407 - Cys 185) between the N- and C- terminal domains (90, 94).

19

Figure 3. Schematic structure of HRG. Dotted & dashed lines represent disulphide bonds, circles with enclosed G represents glycosylation positions. The dotted line (disulphide bond) is located near the HRG C633T SNP. The shaded circle is the glycosylation at position 202 that is present in the HRG isoform with a serine at position 204. N = N-terminal; HRR = histidine-rich region, PRR = proline-rich re-gions; C = C-terminal. Figure adapted from Kassaar et al., 2014.

Gene The HRG gene is located on chromosome 3 at position 3q28 - q29 and con-tains seven exons and six introns (91). The N1 and N2 domains are encoded by three exons. The HRR, PRR and the C-terminal domains are coded by one exon apiece (80). There are ten HRG SNPs identified (www.uniprot.org /uniprot/P04196,2012).

A common SNP in HRG is HRG C633T, which has been assigned the Na-tional Center for Biotechnology Information (NCBI) reference SNP identifi-cation tag rs9898. The HRG gene contains either a cytosine (C633) or thy-mine (633T) at nucleotide position 633. The allele frequency is 0.68 for cy-tosine and 0.32 for thymine in a European population (www.ncbi.nlm.nih. gov/SNP/snp_ref.cgi?type=rs&rs=9898,20140819). A cytosine (C) at nucle-otide position 633 allows for coding of a proline at amino acid position 204 (denoted 186 if the 18 amino acid signal peptide has been omitted) in the HRG protein. A thymine (T) at this position allows for coding of a serine instead. Individuals can be homozygous for the HRG C633 SNP (C/C) and produce only the HRG protein with proline (Pro/Pro) at amino acid position 204; heterozygous for the HRG C633T and produce both HRG with proline and HRG with serine (Pro/Ser); or homozygous for the HRG 633T SNP (T/T) and produce only the HRG protein with serine (Ser/Ser).

The HRG protein containing serine at position 204 has a glycosylation site at position 202. The absence or presence of this glycosylation explains the difference in the molecular weight between the proline (75 kDa) and serine (77 kDa) containing isoforms. The predominant HRG isoform in hu-mans contains a proline. This variant is probably unique to mankind as other species, including primates, only have the isoform containing a serine (www. uniprot.org/uniprot/?query=histidine-rich+glycoprotein&sort=score, 20130826).

20

Functions HRG has been proposed to be an adaptor molecule that interacts with many ligands (Table 1) (95). It also has been described as a protein that regulates various biological systems such as angiogenesis, the immune system, coagu-lation and fibrinolysis and cell survival (apoptosis), (96). A recent study of HRG confirms that HRG functions as an adaptor molecule involved in multi-protein complexes (93).

Table 1. Ligands that interact or bind with HRG

Domain Ligands N-terminal

IgG FcγR C1Q Heparin Heparan sulfate

ATP synthase Necrotic cells Plasminogen Plasmin Thrombospondin

HRR Cations such as Zn2+

Heparin Heparan sulfate

Tropomyosin Microbes Heme

C-terminal

Plasminogen Plasmin

Thrombospondin

Unknown Phospholipids

Heparanase Fibrinogen

DNA Complement compounds

Angiogenesis

Angiogenesis means formation of new blood vessels from pre-existing ones. Through interactions with different ligands, HRG has been suggested to be both pro- and anti-angiogenic (95). The known effects are as follows:

Pro-angiogenic

Blocks thrombospondin’s (TSP) anti-angiogenic effect. TSP binds competitively to the CD36 receptor, blocking fibroblast growth fac-tor-2’s (FGF-2) pro-angiogenic effect. However, the TSP binding area in HRG, located at amino acid region 155-213, is homologous to CD36 and binds with high affinity to TSP, thereby preventing TSP from blocking FGF-2 (97, 98).

Blocks vasculostatin’s anti-angiogenesis effect in a similar manner (86).

Modulates the activity of plasminogen and plasmin, which are in-volved in the remodeling of the ECM. This remodeling is essential for vessel sprouting during angiogenesis (99).

21

Anti-angiogenic

Inhibits endothelial cell proliferation and reduces vascularization (84).

Induces endothelial cell apoptosis. The HRR region of HRG inter-acts with tropomyosin on FGF-2 stimulated endothelial cells and in-duces cell apoptosis (100).

Affects signal transduction targeting focal adhesions or disruption of the structure of the cytoskeleton, thereby interrupting VEGF induced endothelial cell motility (101).

Interrupts tube formation during blood vessel sprouting (101). Reduces FGF’s angiogenic effect by competing with FGF for hepa-

ran sulfate (HS) binding to endothelial cells (102). Prevents the release of angiogenic growth factors bound to HS in the

ECM by blocking the cleavage site on HS or binding to heparanase preventing cleavage of HS (102).

Immune system

HRG affects the immune system by: Prevents the formation of immune complexes and enhances immune

complex solubility and clearance by binding to IgG (103, 104). Regulates the expression and function of FcγR (105, 106). The FcγR

is a receptor found on immune cells such as natural killer cells, mac-rophages, neutrophils and mast cells.

Removes late apoptotic cells through regulation of the FcγR, or by binding directly with the cell’s exposed DNA, intercellular phospho-lipids or cell surface HS (95).

Exhibits antimicrobial characteristics in acidic conditions or by binding with heparin or Zn2+ (107). Hrg knockout mice are more susceptible to bacterial infections (108). HRG is effective against gram positive and gram negative bacteria (109) and has antifungal capacities (110).

Increases or decreases cell adhesion plus interrupts communication and migration to areas of inflammation by immune cells (95).

Coagulation/fibrinolysis system

HRG seems to be involved in both coagulation and fibrinolysis probably as a regulator of these systems. The known effects are as follows:

Decreases coagulation

Interacts with fibrinogen and reduces conversion rate to fibrin (111). Increases pro-thrombin and bleeding time in Hrg +/+ mice (112).

22

Increases coagulation Binds to heparin and decreases deactivation of other coagulation fac-

tors (113).

Decreases fibrinolysis

Interferes with the interaction between plasminogen and fibrin in clots (114).

Decreases fibrinolysis in Hrg +/+ mice (112).

Increases fibrinolysis

Tethers plasminogen to cell surface and increases plasminogen acti-vation (99).

Cell survival and removal of necrotic cells

HRG is involved in cell proliferation, apoptosis and removal of necrotic cells as follows:

Increases smooth muscle cell proliferation (115). Diminishes T-cell (116) and fibroblast proliferation (117). Increases apoptosis and proliferation of tumor cells (84, 118). Interrupts the cell membrane in yeast, causing cell death (110). Facilitates necrotic cell removal (103). Acts as an intermediate between naked DNA and macrophages (119)

and monocytes (104). Binds to exposed cytoplasmic ligands in necrotic cells with permea-

blized cell membranes (104). However, a recent study of HRG puri-fied with a new method, suggests that removal of necrotic cells, may not be through HRG/ligand interactions, but instead due to the activ-ity and/or presence of co-purified molecules in the HRG-complex (93).

Reproduction

Little is known about the function of HRG within the female reproductive tract. Knockout Hrg mice are viable and fertile but have a coagulation defect (112). However, studies of animal reproduction may not apply to humans (120). Mice do not have the HRG isoform that is predominant in humans.

Estrogen is believed to lower the serum concentration of HRG because levels are significantly lower in oral contraceptive users compared to con-trols (121). The plasma level of HRG has been reported to drop to about 50 % of normal levels during the last trimester of pregnancy (122). Interest-ingly, HRG levels in post-menopausal women given estrogen did not change compared with controls (121).

Lindgren and co-workers (123) have found an association with the HRG C633T SNP polymorphism and healthy nulliparous women with recurrent

23

pregnancy losses with an increased proportion of those homozygous for the HRG 633T SNP. Additionally, another HRG SNP (HRG A1042G), known to exert HRG’s anti-angiogenic effect via the HRR region, has been connected with recurrent miscarriage (124).

In women with early-onset preeclampsia, there is an imbalance between placental HRG and fibrinogen, which might contribute to the hypercoagula-bility and the angiogenic imbalance in the placenta (125). HRG levels are also lower in women who developed preeclampsia (126).

Src Homology 2 Domain-Containing Adapter Protein B (SHB) Src homology 2 domain-containing adapter protein B (SHB) is an intra-cellular protein involved in signaling complexes that response to receptor activation. Adaptor proteins lack intrinsic signaling capacity but affect sig-naling through recruitment of other proteins into complexes. SHB was ini-tially discovered in an insulinoma cell line but later found to be expressed universally (127).

Molecular structure The SHB molecule consists of a proline-rich N-terminal domain, a phos-photyrosine binding domain (PTB), four potential tyrosine phosphorylation sites and a Src homology 2 region (SH2) in the C-terminal domain (Figure 4) (128).

Figure 4. Schematic structure of SHB. PTB = phosphotyrosine binding domain, P = phosphorylation sites, and SH2 = Src homology 2 region.

The proline-rich domain allows for SHB to interact with proteins with Src homology 3 domains. There are two known isoforms of the SHB molecule: 64 kDa and 56 kDa. The difference in size is due to five or three proline-rich regions in the N-terminal domain. The PTB and SH2 domains interact with phosphorylated tyrosine in other proteins. The tyrosine phosphorylation sites in SHB allow for interactions with PTB or SH2 domains in other pro-teins. The ligands or receptors SHB is known to interact with are found in Table 2 (128).

24

Table 2. SHB ligands and function

Domain Ligand/receptor Function Proline-rich SRC Angiogenesis GRAP, GADS, PLC-γ, JAK 1/3 T-cell signaling GRB2

T-cell signaling β cell proliferation and survival

c-ABL Apoptosis in oxidative stress EPS8 Cytoskeletal dysfunctions PTB LAT, VAV-1 T-cell signaling FAK Neurite outgrowth regulation Tyrosine-P RAS-GAP Angiogenesis, apoptosis, SH2 Angiogenesis, neuritogenesis, SLP76 T-cell signaling CRK Neurite out-growth regulation SH2 FGFR, VEGFR-2 Angiogenesis IL-2 receptor Apoptosis PDGFR Cytoskeleton in fibroblasts Unknown SHP-2 Angiogenesis, apoptosis, IRS-2 Beta cell proliferation

This table exemplifies the many ligands, receptors and biological systems affected. PTB = Phosphotyrosine binding domain; Tyrosine-P = Tyrosine phosphorylation; GRAP = GRB2 (growth factor receptor-binding protein)-related adapter protein; GADS = GRB2-related adapter downstream of SHC; PLC-γ = Phospholipase; JAK = Janus kinase; GRB = Growth factor receptor-binding protein; c-Abl = Abelson murine leukemia viral oncogene homolog 1; EPS8 = Epidermal growth factor recep-tor kinase substrate 8; LAT = Linker for activation of T cells; FAK = Focal adhesion kinase; RAS-GAP = Rat sarcoma-GTPase; SH2 = SRC homology 2; SLP-76 = SH2 domain containing leukocyte phosphoprotein of 76 kDa; CRK = CT10 regulator of kinase; FGFR = Fibroblast growth factor receptor; VEGFR = Vascular endothelial growth factor receptor; IL = Interleukin; PDGF = Platelet derived growth factor; SHP-2 = SH2-containing tyrosine phosphatase; IRS = Insulin receptor substrate.

Gene The SHB gene is located on chromosome 9 at locus p12 - p11 (http://www-ncbi-nlm-nih-gov.ezproxy.its.uu.se/gene/6461). It is 150 000 base pairs long and includes six exons. Exon 1 codes for the proline-rich and PTB regions. Four potential tyrosine phosphorylation sites are found within exons 2 and 3 and exons 5 and 6 code for the SH2 region (128).

Functions SHB is involved in many biological systems such as angiogenesis, the im-mune system and apoptosis. SHB has pleiotropic effects and the stimulatory

25

factor and the cell type involved will determine its effect (Table 2) (129). Through its close interaction with FRK/RAK, it may be important in tumor suppression. The known effects are as follows:

Angiogenesis

• Enhances the effect of angiogenesis inhibitors through increased en-dothelial cell apoptosis (130).

• Decreases tube cell formation (131). • Affects the cell cytoskeleton of endothelial cells (131). • Relays signals from activated FGFR-1 (132) and from activated

VEGR-2 (133) in endothelial cells. • Influences the migration of endothelial cell stimulation by FGF-2

(134). • Regulates focal adhesions and cell migration of endothelial cells ac-

tivated by VEGF through the VEGFR-2 (133). • Impacts tumor cells angiogenesis (135).

Immune system

• Associates with the CD3 complex in T-cell receptor signaling and is important for Il-2 secretion (136, 137).

• Interacts with the IL-2 receptor signaling in T-cells and natural killer cells (NK) (137).

• Influences hematopoietic differentiation in embryotic stem cells (138).

• Affects how CD3 and CD28 stimulate T lymphoid cells (139).

Apoptosis

• Overexpression of SHB increases apoptosis in fibroblasts (140), en-dothelial cells (130, 131, 134), beta cells (141) and prostate cancer cell (142).

Reproduction

Knowledge of SHB’s effect on reproduction has been gained through Shb knockout mice studies (143). The Shb gene knockout mice are viable and fertile but no Shb -/- pups have been obtained when mating Shb null mice with heterozygous mice (143).

The Shb +/- mice ovulate Shb - oocytes more often (143), though no ex-planation has been given. Shb knockout mice have accelerated oocyte devel-opment until one week post-natally. However, in the adult, the ratio of pri-mary or antral follicles to primordial follicle ratio is lower for the knockout mice. The completion of meiosis I is also less synchronized compared with wild-type mice. Some oocytes display accelerated meiosis and others, re-tarded meiosis (144).

26

Shb knockout mice have impaired early embryo development in the -/- embryos (144). Fewer Shb -/- embryos reached the morula stage and those reaching blastocyst stage were less mature or had inner cell mass and trophectoderm abnormalities. Even embryonic stem cell studies have altered differentiation patterns in mesodermal and endodermal cell lines between those lines deficient in SHB and normal cell lines (145). Thus, SHB seems to affect early embryonic development.

The Shb - allele is associated with malformation and resorption of pups. Many of the early losses of Shb -/- embryos occurred before the stage of resorption, thus, at or before early implantation. Due to the wide range of birth defects, it has been suggested that SHB affects all stages of fetal devel-opment (143). Knockout mice have impaired angiogenesis (135), glucose homeostasis (146) and beta cell response to stress (147) so these may be the pathophysiological mechanisms affecting fetal development.

There are no previous fertility studies of SHB in humans nor are there any studies of the effect of different SHB SNPs on fertility.

27

Aims

The main aim of this thesis was to investigate the impact of HRG and SHB on human female fertility by achieving the following specific research objec-tives: I: To investigate the presence, distribution and expression of HRG in the human female reproductive tract, placenta and embryos. II: To investigate if the single nucleotide polymorphism HRG C633T is as-sociated with pregnancy results in IVF. III: To investigate if the HRG C633T SNP is associated with ovarian re-sponse and embryo quality in IVF. IV: To investigate the association between different single nucleotide poly-morphisms of the SHB gene and prognostic markers associated with preg-nancy outcome for women undergoing IVF.

28

Material and methods

Paper I and II

Study participants

Infertile women Women (n = 24), undergoing IVF at the Reproduction Center, Uppsala Uni-versity Hospital, were recruited (Paper I and II, cohort 1, Table 3). Inclusion criteria were: age < 40 years, BMI < 35 kg/m2, bFSH (day 2 – 5) ≤ 10 IU/l and not more than two previous IVF treatments. Median age was 32.0 years, median BMI was 23.9 kg/m2 and 23.8 % were smokers.

To validate the results from cohort 1 (Paper II), a second cohort (cohort 2, Table 3) was included. This group consisted of women diagnosed with un-explained infertility (n = 34) undergoing a first or second IVF at the Fertility Unit, Karolinska Hospital Huddinge, Stockholm. All participants were < 39 years old, had a BMI ≤ 32 kg/m2 and bFSH serum concentration ≤ 13 IU/l.

Table 3. Summary of participant groups

Paper I, II Cohort 1

II Cohort 2

III Cohort 3

IV Cohort 3

IV Control

Sample

Infertile women

Infertile women

Infertile women

Same as Paper III

Pregnant women

Participants Recruited (n)

24 36 155 Same as Paper III

100

Subgroup

Western Blot

successful

SNP analysis

successful

SNP analysis

successful

SNP analysis

successful

SNP analysis

successful Analyzed Subgroup Diagnosis

Various infertility

causes

Unexplained infertility

Unexplained infertility

Variousinfertility

causes

Healthy, normal

pregnancy Analyzed Subgroup (n)

24 36 67 140 98

29

Methods

Western blot on follicular fluid and plasma Plasma and follicular fluids were collected at OPU (cohort 1). Western blot was performed using an anti-HRG antibody directed towards the His/Pro-rich domain of HRG (HRG-0119). Immunoreactive sites were detected by the fluorescently labeled anti-rabbit IRDye800 antibody.

ELISA on culture medium Enzyme-linked immunosorbent assay (ELISA) was used to analyze HRG levels in medium used for human blastocyst culture. Plates coated with a HRG capture antibody (H00003273-B01P, AbNova, Germany) were used. The affinity-purified rabbit anti-HRG antibody (HRG-0119) and a biotinyl-ated anti-rabbit immunnoglobulun G antibody (BA-1000, Vector Laborato-ries, USA) were used for detection.

Immunohistochemical staining of embryos Donated human embryos of quality not fit for transfer or cryopreservation were used for immunohistochemical staining. This was performed in accord-ance with standard procedures with affinity-purified rabbit anti-HRG anti-body (HRG-0119) as the primary antibody and Alexa 568 conjugated goat anti-rabbit (A11079) as the secondary antibody. DAPI (4’,6-diamidino-2-phenylindole) was used to detect the locations of nuclei.

Immunohistochemical staining of tissue Fallopian tube and endometrial biopsies were obtained from a separate group of healthy, fertile women undergoing sterilization. Tube biopsies were from all phases of the menstrual cycle. Endometrium samples were taken six days post-ovulation. Placenta and uterine myometrium biopsies were collected at Caesarean section from a new group of women with normal pregnancies.

The biopsies were embedded in paraffin wax in accordance with routine procedures. Sections of Fallopian tube, endometrium, placenta or myometri-um were incubated with the HRG-0119 antibody and a biotinylated anti-rabbit antibody. Each section was counterstained with Mayer´s hematoxylin.

Real-time PCR (RT-PCR) on tissues and embryos After preparation of RNA from endometrium, Fallopian tube, placenta, my-ometrium biopsies and blastocysts (n = 8), cDNA was synthesized and RT-PCR reactions were performed in accordance with standard protocol. As an endogenous control, the target genes were normalized to glycerylaldehyde-3-phosphate dehydrogenase (GAPDH).

30

Preparation of DNA and HRG C633T SNP analysis DNA was extracted from blood samples using QIAamp DNA Blood Maxi kits (Qiagen, Venlo, The Netherlands). Each DNA sample was genotyped for the rs9898 C/T (HRG C633T) SNP in HRG exon 5, using the TaqMan SNP Genotyping Assay (Applied Biosystems, Foster City, CA, USA). Re-sults were correlated to pregnancy test results.

Paper III and IV

Study participants Infertile women A new group of women (cohort 3, n = 155, Table 3) with diverse causes of infertility undergoing IVF were recruited at Fertilitetscentrum Stockholm between March 2010 and February 2012. All had been screened for infertili-ty cause. The infertility causes were: anovulation (13.2 %); tubal (11.1 %); male factor (25.7 %); unexplained (47.9 %); endometriosis (6.3 %); and other (3.5 %). Women with multiple causes of infertility are included above (7.6 %). Median age was 34.0 years, median BMI was 23.0 kg/m2, percent-age smokers was 11.9 %, median menstrual cycle length was 28.9 days and median duration of infertility was 2.5 years. Data was obtained from medical records. IVF treatment type and doses were determined individually based on anticipated response and previous treatment results.

Fertile, pregnant women A control group of 100 women with normal fertility was recruited at Uppsala University Hospital at a screening ultrasound in the 16 - 20th week of preg-nancy (Paper IV, Table 3). The median age was 31.0, median BMI (first trimester) was 23.9 kg/m2, 2.0 % were smokers, median menstrual cycle length was 28.0 days, median number of previous pregnancies was two and median parity at recruitment was one. Data was obtained from medical rec-ords. Participants had no history of infertility, medical or pregnancy prob-lems. Women who developed complications were excluded (n = 2).

Methods Venous blood samples from infertile (cohort 3) and fertile women were col-lected. DNA was extracted from buffy coat and genotyped for the HRG C633T SNP as described previously (Paper II). DNA samples were also sent to KBioscience (Hoddesdon, UK) for SHB SNP genotyping (Paper IV). The following SHB SNPs were analyzed: rs2025439; rs13298451 and rs7873102.

31

Women were grouped for each SHB SNP as homozygous for the major allele (1/1), heterozygous (1/2) or homozygous for the minor allele (2/2).

Results were analyzed together with IVF parameters obtained by exami-nation of medical records. Genotype was determined after pregnancy result and thus blinded for treating physician and embryologist. Embryos were assessed in accordance with standard clinical morphological scoring (53).

Nomenclature of HRG polymorphism The same HRG SNP has been studied: rs9898 C/T (Paper II) and HRG C633T (Paper III). For simplicity in Paper II, groups were named according to the protein isoform(s) produced. Those homozygous for the major allele (HRG C633) have been classified in Paper II according to protein isoform (Pro/Pro) and in Paper III according to the SNP nucleotide (C/C). Heterozy-gous (HRG C633T) have been classified in Paper II as Pro/Ser and in Paper III as C/T. Those homozygous for the minor allele (HRG 633T) have been classified in Paper II as Ser/Ser and in Paper III as T/T.

Statistical analysis Statistical analysis was performed using the Statistical Package for Social Sciences, versions 15.0 and 20.0 (SPSS, IBM Corp., USA). Differences in categorical data proportions were analyzed using a Pearson’s chi-squared test. For expected frequencies less than five or if any frequency was less than one, a Fischer’s exact test was used (Papers II, III and IV). Odds ratios (OR) with 95 % confidence interval (CI) were obtained using bivariate logistic regression (Paper II). Medians with interquartile range were used for ordinal or continuous data with a limited sample size. A Mann-Whitney U test was used for comparison of genotypes (Papers III and IV). Graphical analysis and Pearson’s correlation coefficient were used in sub-group analysis to study correlation between continuous variables (Paper IV).

Haploview software (www.broad.mit.edu/mpg/haploview) was utilized to investigate departures from Hardy-Weinberg equilibrium (HWE) and to assess linkage disequilibrium (LD). This software was also used for haplo-type reconstruction from the genotype data (Paper IV).

Statistical tests were two-sided and p ≤ 0.05 was considered significant.

Ethical Approval All studies were approved by the Regional Ethics Committee in Stockholm and/or Uppsala. Informed consent was obtained from all participatants.

32

Results

Paper I The presence and expression of HRG in the female reproductive tract and embryos were studied.

HRG in follicular fluid Western blot analysis of samples of follicular fluid (n = 24) and plasma (n = 24) demonstrated that HRG migrates as two bands of different molecu-lar weights (75 and 77 kDa) corresponding to the two HRG isoforms. All participating women were found to have the same HRG isoform(s) in follicle fluid as in plasma.

HRG in culture medium and embryos ELISA was performed on medium from blastocyst culture to analyze if HRG is secreted by human embryos. HRG was detected in all samples. Control medium did not contain a detectable level of HRG.

To study the presence, localization, and intensity of HRG in embryos, immunohistochemical staining was performed. HRG was found at all stages of development. HRG was detected in cytoplasm of all cells regardless of developmental stage. However, there were more blastocyst with HRG found in nuclei (n = 17/18, 94 %) than cleavage stage embryos (n = 4/16, 25 %; p < 0.001). None of the embryos exhibited HRG in all cell nuclei. RT-PCR analysis was performed to investigate if HRG was expressed by the embryos. The mean CT value for HRG was 35.85 ± 1.89 and for GAPDH 26.26 ± 0.76, which is close to the detection limit for HRG mRNA.

HRG in the female reproductive tract Immunohistochemical staining was used to detect the presence of HRG in the female reproductive tract. HRG was found in most cell types. HRG was observed in human endometrium, Fallopian tube and in myometrium. Addi-tionally, in the placental tissue, HRG was found in endothelial, stromal, and trophoblast cells. No mRNA expression of HRG in tissues from Fallopian tube, endometrium, placenta or myometrium was detected using RT-PCR.

33

Paper II The effect of the HRG C633T SNP on pregnancy results in IVF was studied in this paper.

Pregnancy results related to HRG protein isoform or genotype Individual pregnancy results (cohort 1) were analyzed in relation to Western blot results (Table 4). The majority of those who became pregnant were Pro/Pro. None of the Ser/Ser women became pregnant. The results of the HRG C633T SNP analysis (cohort 2) were similar to the Western blot results (Table 4). The results from combining the cohorts (cohorts 1 and 2) found that 26 women became pregnant, of which 21 (n = 21/26, 80.8 %) were Pro/Pro. The remaining five (5/26, 19.2 %) who became pregnant were het-erozygous.

Table 4. Pregnancy results in relation to HRG protein isoform or SNP

Method Genotype

Pregnant Not Pregnant

Total Ratio pregnant

Western blot Pro/Pro 8 (88.9) 6 (40.0) 14 (58.3) 0.571 Pro/Ser 1 (11.1) 5 (33.3) 6 (25.0) 0.167 Ser/Ser 0 (0) 4 (26.7) 4 (16.7) 0 Total 9 15 24 0.375 SNP analysis Pro/Pro 13 (76.5) 5 (29.4) 18 (52.9) 0.722 Pro/Ser 4 (23.5) 10 (58.8) 14 (41.2) 0.286 Ser/Ser 0 (0) 2 (11.8) 2 (5.9) 0 Total 17 17 34 0.500 Combined cohorts Pro/Pro 21 (80.8) 11 (34.4) 32 (55.2) 0.656 Pro/Ser 5 (19.2) 15 (46.9) 20 (34.5) 0.25 Ser/Ser 0 (0) 6 (18.8) 6 (10.3) 0 Total 26 32 58 0.448 Allele frequencyCombined cohortsa Pro 47 37 84 0.560 Ser 5 27 32 0.156

Values are n (%). ap < 0.05 when testing the null hypothesis that allele frequencies are equally distrib-uted in the two groups. Pro/Pro = homozygous for the HRG C633 SNP; Pro/Ser = heterozygous for the HRG C633T SNP; Ser/Ser = homozygous for the HRG 633T SNP.

34

Paper III The association between the HRG C633T SNP and demographic, treatment and laboratory variables in IVF were studied in this paper. The criteria for exclusion were limited in order to enable investigation of a broad patient group, but also to facilitate sub-group analysis. Investigations could then determine if there were differences in predominate genotypes compared with other groups or if a genotype within a sub-group affected IVF variables. The HRG C633T SNP was strongly associated with unexplained infertility and therefore, the analysis was focused on this sub-group of 67 women whose DNA was successfully analyzed.

Demographic variables The distribution of the HRG C633T genotype in this paper (homozygous for the HRG C633 SNP (C/C): 58.2 %; heterozygous for the HRG C633T SNP (C/T): 34.3 %; homozygous for the HRG 633T SNP (T/T): 7.4 %; p > 0.05) was similar to that previously described in a European population (C/C: 47.2 %; C/T: 41.7 %; T/T: 11.1 %) (www.ensembl.org/Homo_sapiens/Variation /Population?db=core;r=3:186390127186391127;v=rs9898;vdb=variation;vf=31250, 140307). No differences were found between genotype groups regarding age, BMI, percentage of current smokers, menstrual cycle length, bFSH, AMH or AFC. Similar to the results in Paper II, there was a tendency for the T/T group to have a lower percent of women who had a previous pregnancy or child compared with those in the C/C or the C/T group (Table 5).

Table 5. Previous pregnancy stratified by HRG C633T SNP

HRG C633T SNP C/C C/T T/Tn (%) 39 (58.2) 23 (34.3) 5 (7.5) Pregnancya 13 (33.3) 12 (52.2) 1 (20.0) Miscarriagea 7 (17.9) 7 (30.4) 1 (20.0) Childa 4 (10.2) 0 (0) 0 (0)

anon-significance.

Treatment background/parameters There were no differences between the SNP groups regarding the treatment number, method used (IVF, ICSI or combined), treatment protocol type (agonist or antagonist), type of FSH (rFSH or hMG) or OPU day. Significantly lower total dosages of FSH were administered to women in the C/C group than the other groups combined (p < 0.05) (Table 6).

35

Laboratory parameters The median number of oocytes obtained was lower for the T/T group than the other groups. The T/T group also received fewer mature oocytes. Moreo-ver, the number of fertilized oocytes was lowest in the T/T group (Table 6) and there was a tendency for this group to have a lower fertilization rate. The T/T group had the highest percent (40 %) (C/T: 17.4 %; C/C: 5.1 %) of women whose embryos were entirely unfit for transfer although this differ-ence was not significant (p = 0.06).

Table 6. IVF variables stratified by HRG C633T SNP

C/C 39 (58.2)

C/T 23 (34.3)

T/T 5 (7.5)

FSH/hMG (IU/L) 1875 (1350 – 2250)a

1650 (1250 – 3000)a

3150 (1363 – 6038)a

Oocytes 8 (6.0 – 13.0)b

8 (6.0 – 11.0)b

5 (3.0 – 6.5)b

Mature oocytes 8 (5.0 – 12.0)b

7 (5.0 – 9.0)b

4 (2.0 – 5.5)b

Fertilized oocytes 6 (3.0 – 9.0)c

5 (3.0 – 7.0)c

2 (2.0 – 4.0)c

Fertilization rate (%) 64.3 (50.0 – 87.5)d

64.3 (42.9 – 77.8)d

33.3 (12.5 – 65.7)d

No ET n (%) 2 (5.1)e 4 (17.4)e 2 (40.0)e

Data are median (interquartile range) unless otherwise stated. No ET = proportion of women who had exclusively poor-quality embryos unfit for transfer. ap < 0.05 when comparing C/C with the other groups combined, bp < 0.05 when comparing C/C to T/T, C/T to T/T and T/T with the other groups combined, cp < 0.05 when comparing C/C with T/T and comparing T/T with other groups com-bined, dNon-significant, p = 0.08 when comparing T/T with other groups combined; ep = 0.06 when comparing C/C to T/T.

Paper IV The association between three SHB SNPs and demographic, treatment and laboratory variables in IVF were studied in this paper.

Allele frequencies, haplotypes and linkage disequilibrium The following SHB SNPs were successfully analyzed: rs2025439 (n = 140 IVF; n = 97 controls), rs13298451 (n = 140 IVF; n = 98 controls) and rs7873102 (n = 139 IVF; n = 98 controls). For all SHB SNP groups, the ma-jor allele had a guanine at the SNP position. The minor allele had either an adenine (rs2025439, rs13298451) or thymine (rs7873102) at their respective SNP position. The SHB SNP allele frequencies are presented in Table 7. The

36

frequencies are similar to those previously described in a European popula-tion (http://www-ncbi-nlm-nih-gov.ezproxy.its.uu.se/projects/SNP/snp_ref. cgi, 140524). All SNPs were in Hardy-Weinberg equilibrium (rs2025439, p = 0.72; rs13298451, p = 0.85; rs7873102, p = 0.52).

Table 7. SHB allele association for different SNPs

Infertile Fertile

SNP Major:Minor allele (n)

Major (%) Major:Minor allele (n)

Major (%)

rs2025439a 227:53 81.1 152:42 78.4 rs13298451a 235:43 84.5 157:39 80.1 rs7873102a 161:117 57.9 104:92 53.1

aNon-significant difference for each SNP group when comparing infertile and fertile groups.

The predicted inheritance patterns of haplotypes are presented in descending order in Table 8. There was no difference between the infertile and fertile groups regarding allele frequencies (Table 7) or the most commonly inherit-ed SHB haplotypes (Table 8). The interactions between all three SNP pairs have been determined to have a strong LD (rs2025439 and rs13298451, LD = 92 %; rs13298451 and rs7873102, LD = 90 %; and rs2025439 and rs7873102, LD = 81 %).

Table 8. The most common SHB haplotypes for the SNPs studied

Haplotype Total (%) Infertile (%) Fertile (%)

111a 53.3 55.5 50.1

112a 25.8 25.5 26.4

221a 15.7 14.7 17.2

211a 1.8 2.1 1.4

212a 1.5 1.1 2.1

1 = major allele; 2 = minor allele. Within the haplotype block, the first number cor-responds to rs2025439, the second number to rs13298451 and the third number to rs7873102. aNon-significant difference for each haplotype when comparing infertile with fertile groups.

Demographic variables Infertile women Age, percentage of smokers, menstrual cycle or infertility length, bFSH, AMH and AFC were similarly distributed between for genotypes within each SNP group (Paper IV, Supplementary Table 1). A higher median BMI

37

(23.4 kg/m2) was found for the 1/2 group (rs7873102) compared with the other groups (1/1: 22.7 kg/m2; 2/2: 22.0 kg/m2).

Fertile women A difference in BMI was found for rs13298451 group. The highest median BMI was in the 2/2 group (27.8 kg/m2) compared with the 1/1 group (23.4 kg/m2) and the 1/2 group (24.3 kg/m2, p < 0.05 when 2/2 was compared with 1/2).

Treatment parameters No differences between genotypes were found for the infertile women within each SNP group regarding the proportion of IVF, ICSI or combined (IVF and ICSI) treatments, the proportion of agonist or antagonist treatments or the median OPU day (Paper IV, Supplementary Table 2). The current treat-ment number was lower for the rs13298451 1/2 group compared with the 1/1 group or with both of the other groups combined. For the rs13298451 group, rFSH was given more often to the 1/1 and hMG more often to the 2/2 group (Paper IV, Supplementary Table 2). Within the rs13298451 group, the high-est median amount of total gonadotropins was given to the heterozygous group. For the rs7873102 group, the 1/1 genotype group received the highest total gonadotropin dosages (Table 9).

Laboratory parameters Oocyte number, maturity and fertilization The 2/2 group had fewer fertilized oocytes (rs2025439 and rs13298451, Table 9) due to fewer oocytes obtained and not because of a difference in fertilization rate (Paper IV, Supplementary Tables 3A-C). Contrastingly, for rs7873102 the number of oocytes was similar among different genotypes (Paper IV, Supplementary Table 3 A-C). However, for this SNP (rs7873102) the 1/2 group had a higher percentage of immature oocytes obtained per woman (Table 9).

The 1/2 group (rs7873102) was associated with both a higher BMI and a higher percentage of immature oocytes. To test if the difference in the per-centage of immature oocyte was dependent on the differences in BMI, the results were graphed and a correlation coefficient was calculated. No signifi-cant correlation was found between these two variables (p = 0.90), which indicates that this SNP affected BMI and the percentage of immature oocytes independently.

38

Embryo quality and day of transfer The 2/2 group had fewer good-quality embryos (rs13298451,Table 9). The 1/2 group and the 2/2 group underwent ET more often on day 2 - 3 and the 1/1 underwent ET more often on day 4 – 5 (rs7873102, Table 9). This was seen as a tendency in the other SNP groups (Paper IV, Supplementary Tables 3 A-C).

39

Tab

le 9

. IV

F v

aria

bles

str

atif

ied

by S

HB

SN

P

S

NP

1/

1 1/

2 2/

2 F

SH

(IU

/L)

rs13

2984

51a

1575

(13

50 –

180

0)

1750

(13

50 –

256

6)

1500

(11

62 –

181

3)

rs

7873

102a

1875

(15

00 –

260

0)

1687

(12

38 –

225

0)

1500

(12

00 –

173

7)

Fer

tiliz

ed

rs20

2543

9b 5.

5 (3

.0 -

8.3

) 4.

0 (2

.0 -

8.0

) 2.

0 (1

.0 -

3.0

)

rs13

2984

51b

5.0

(3.0

- 9

.0)

4.0

(2.0

-7.

0)

1.0

(1.0

-)

Imm

atu

re (

%)

rs78

7310

2c 0.

0 (0

.0 –

14.

3)

12.5

(0.

0 –

23.2

) 0.

0 (0

.0 –

19.

4)

GQ

E (

%)

rs13

2984

51d

25.0

(12

.5 –

42.

9)

33.3

(16

.2 -

50.0

) 8.

3 (0

- )

E

T d

2 -

3 n

(%

) rs

7873

102e

33 (

80.5

) 53

(94

.6)

23 (

100)

E

T d

4 -

5 n

(%

) rs

7873

102e

8 (1

9.5)

3

(5.4

) 0

Cry

opre

serv

ed d

2 -

3

rs13

2984

51f

0.5

(0.0

- 2

.0)

2.0

(0.0

- 4

.0)

0

Dat

a ar

e m

edia

n (i

nter

quar

tile

ran

ge)

unle

ss o

ther

wis

e sp

ecif

ied.

FS

H =

tota

l gon

adot

ropi

n do

sage

; Fer

tiliz

ed =

tota

l num

ber

fert

ilize

d; I

mm

a-tu

re =

num

ber

of M

I +

Gv

oocy

tes

per

tota

l oo

cyte

and

wom

an;

GQ

E =

num

ber

of g

ood-

qual

ity

embr

yos

per

tota

l nu

mbe

r of

ooc

ytes

and

w

oman

; E

T d

2 -

3 =

pro

port

ion

with

ET

day

2 o

r 3;

ET

d 4

- 5

= p

ropo

rtio

n w

ith E

T d

ay 4

or

5; 1

/1 =

hom

ozyg

ous

for

maj

or a

llele

; 1/

2 =

he

tero

zygo

us; 2

/2 =

hom

ozyg

ous

for

min

or a

llele

.

a FS

H:

rs13

2984

51:

p =

0.0

3 w

hen

1/1

com

pare

d w

ith 1

/2 a

nd p

= 0

.03

1/2

com

pare

d w

ith t

he o

ther

gro

ups

com

bine

d; r

s787

3102

: p

= 0

.01

whe

n co

mpa

ring

1/1

with

2/2

and

p =

0.0

3 w

hen

com

pari

ng 1

/1 w

ith th

e ot

her

grou

ps c

ombi

ned.

b F

erti

lized

: rs

2025

439:

p

=

0.02

w

hen

1/1

com

pare

w

ith

2/2,

p

=

0.03

w

hen

2/2

com

pare

d w

ith

the

othe

r gr

oups

co

mbi

ned;

rs13

2984

51: p

= 0

.03

whe

n 1/

1 co

mpa

re w

ith 2

/2 a

nd p

= 0

.04

whe

n 2/

2 co

mpa

red

with

the

othe

r gr

oups

com

bine

d.

c Imm

atu

re: p

= 0

.04

whe

n 1/

1 co

mpa

red

with

1/2

and

p =

0.0

3 w

hen

1/2

com

pare

d w

ith th

e ot

her

grou

ps c

ombi

ned.

d G

QE

: p =

0.0

4 w

hen

1/2

com

pare

d w

ith 2

/2.

e ET

(d

ay 2

- 3

com

pare

d w

/d 4

– 5

): p

= 0

.05

whe

n 1/

1 co

mpa

re w

ith 1

/2 a

nd p

= 0

.04

whe

n 1/

1 co

mpa

re w

ith 2

/2.

f Cry

opre

serv

ed: p

= 0

.04

whe

n 1/

1 co

mpa

red

with

1/2

.

40

Discussion

Methodological considerations Human fertility is difficult to study. Studying fertile couples can be poten-tially impractical, subject to recall bias and/or unethical. Often infertile cou-ples are studied instead. These couples represent a heterogeneous group with various underlying causes of infertility, some of which may not be detected. Also, this group includes fertile patients. Longitudinal studies of infertile couples are time consuming and complicated by patients switching treatment types or having differing time periods between treatments.

Biomolecular aspects of reproduction can be studied in the laboratory by animal or in vitro experiments. However, when animal models have been used, there are often problems extrapolating the results to human conditions, even when working with primates (120), because implantation, placentation, pregnancy length and embryo development differ between species. For the same reasons, in vitro models, such as cell cultures, cannot fully mimic the complexity of human biology.

The studies within this thesis are mainly performed with a translational approach, which is a strength and quite unique. The study designs chosen have been cross sectional and are of a pilot-study nature. HRG is studied from a variety of perspectives, such as its presence or expression in embyos/tissues (Paper I) and its effect on IVF variables (Paper II and III). The study of SHB (Paper IV) is a continuation of a pre-clinical study of knockout mice. A strength of the study is that all fertile women included had proven and not assumed fertility (Paper I, IV). It is both a strength and a weakness that there were few exclusion criteria for participation within the infertile cohorts (Paper II, III and IV). This means that the patients included probably represent those met in a clinical setting, not just a selected group. However, comparisons become more difficult because of the heterogeneity of participants. Ethnicity is not always easy or ethical to assess (148). There-fore this thesis has not examined ethnical differences (Paper II, III and IV). The inclusion frequency was high and the population studied in Paper III and IV is well defined. It is a strength that the results are similar (Paper II and III) despite cohorts from three different clinics, each having different treat-ment and laboratory routines.

Paper I has been limited to sub-optimal quality embryos because it would have been unethical to use embryos desired for potential pregnancies. How-

41

ever, knowledge gained from these embryos may help understanding about variations in embryo quality. Assessment of embryo quality has been limited to morphological evaluation (Paper III and IV) as this is the most precise tool currently in use in Sweden. Morphological assessment is dependent upon the knowledge and experience of the embryologist, although guideline have improved standardization (53). A strength of these studies is that em-bryo assessment has been performed by different embryologists thereby decreasing inter-individual differences.

A limitation is that the whole female reproductive tract has not been stud-ied. However, it is a strength that we have studied all areas where the oocyte develops, is fertilized and later implants and develops. A further strength is that both the presence and expression of HRG have been studied (Paper I).

Potential limitations, such as errors in self-reported data (149, 150), recall bias or regional differences in routines or laboratory analysis methods fol-lowed to diagnose infertility are spread evenly among genotypes (Paper II, III and IV). Genotype was blinded for personnel and participants, which is a strength, because prior knowledge of genotype did not affect any of the vari-ables studied (Paper II, III and IV).

HRG and female fertility

Ovaries and oocytes The results, showing that the HRG C633T SNP is associated with the dosag-es of gonadotropin given, the number of oocytes retrieved and the number fertilized during IVF, is interesting. In a clinical setting, it is difficult to get an actual count of the number of oocytes remaining and therefore, indirect variables are used, such as bFSH, AMH or AFC (49). Because the age, bFSH, AMH and AFC did not differ; the HRG C633T SNP probably does not affect the number of remaining oocytes. However, it might affect oocyte quality. One would have expected that the HRG C633T SNP should affect the ovarian reserve because HRG is involved in angiogenesis and apoptosis, both systems involved in the development and maintenance of the oocyte pool (20, 151). However, all of the above measures comprise of indirect indications of the oocyte pool and further studies using ovarian biopsies could elucidate this discrepancy.

FSH threshold levels for the recruitment of follicles are individual due to factors such as age, polymorphisms in the FSH receptor (77), FSH metabolic clearance and ovarian sensitivity (152-154). Women with high threshold levels have poorer pregnancy chance (155). However, even within this group, the chance of pregnancy can vary. Other factors associated with preg-nancy chance are the number of oocytes retrieved and the number of ferti-

42

lized oocytes (156-158). The studied HRG genotype affects the number of fertilized oocytes. Women given lower gonadotropins dosages obtained the most fertilized oocytes, indicating that the HRG C633 group’s ovaries are more sensitive to gonadotropins than the HRG 633T group. Because not all follicles contain oocytes, it would have been interesting to investigate if the number of mature follicles differed between genotype groups. The number of immature oocytes and the percentage of immature oocytes did not differ based on HRG genotype. This implies that the HRG C633T SNP does not appear to affect oocyte maturity.

Oocytes communicate with cumulus cells via paracrine and gap-junctional signaling (22) and will not develop if this communication is inter-rupted (23, 24). An association between gene expression and chromosome abnormalities in oocytes has been reported (159). A study of cumulus cells gene expression found differences in the expression of genes linked with embryo development for genes involved in angiogenesis, apoptosis, various growth factors signaling (including FGF), general vesicle transport and chemokine/cytokine signaling (160). A gene expression study of human cumulus cells from MI respectively MII oocytes did not report any differ-ence in HRG expression (161) However, it might be possible that HRG could influence oocyte/cumulus communication through altered expression of HRG ligands.

Follicular fluid The finding that HRG exists in follicular fluid has later been confirmed (162). The HRG found in follicular fluid may reflect an ongoing angiogenic process in the development of the follicle and the oocyte. Heparin-binding angiogenic factors, such as FGF and VEGF, are produced by the pre-ovulatory follicle and regulate the angiogenic balance in the follicle (163-165). HRG interacts directly and indirectly with angiogenic, antiangiogenic factors and heparin (79) and might be important for follicle development. High VEGF levels in follicular fluid are associated with poor oocyte quality (166), poor fertilization (167) and poor-quality embryos (168). A study of the correlation between follicular fluid content and the fertilization or non-fertilization of the corresponding oocyte revealed differences in expression of the heparan sulfate proteoglycan perlecan gene (169). HRG’s interaction with HS is known to be of relevance in angiogenesis (170). Another mecha-nism might be through microvesicle in follicular fluid. These contain biolog-ically active material and are involved in oocyte/cumulus communication. A study (171) found a HRG-like protein in microvesicles in mares. If HRG in follicular fluid regulates angiogenic factors or functions via microvesicles, it may also be of importance for the development of the oocyte.

43

Fertilization The difference in the number of fertilized oocytes probably reflects the total number of oocytes obtained. However, there was a tendency for the group with fewer oocytes to have a lower fertilization rate. There are no studies of HRG and fertilization. A study of Hrg knockout mice (112) found that mating of heterozygous adults yielded an approximate average of nine pups per mating, although the proportions of homozygous and heterozygous pups obtained (Hrg +/+ 30.6 %; Hrg +/- 34.7 %; and Hrg -/- 34.7 %) differed from the expected ratios (+/+ 25 %; +/- 50 %; -/- 25 %).

Embryo development and implantation/placentation The number of good-quality embryos is associated with chance of live birth (156, 157, 172). Therefore, it is not surprising that the genotype group that had the lowest pregnancy chance also had the highest proportion of women having only embryos unfit for transfer. All embryos were in the cleavage stage, suggesting that the poor embryo quality might be due to oocyte quality. Cleavage at this stage is not dependent upon the embryo’s own genome, which is activated after the 4 - 8 cell stage (31). The quality of oocytes is difficult to assess morphologically (52). Instead, the number of good- quality embryos might be suggested as a proxy for oocyte quality.

HRG is located in the blastocyst nucleus, in medium from blastocyst culture and in mRNA in the blastocyst, which suggests that the embryo produces HRG. The results presented in this thesis are the first indicating that blastocysts produce HRG. However, due to ethical considerations, sub-optimal embryos were used. Taking into account the association with HRG and apoptosis, it cannot be excluded that the HRG produced by the embryo is due of apoptosis. Further studies on good-quality embryos such as those donated for research are needed.

The HRG produced by blastocysts might be of importance in the various steps regulating implantation and placentation. A number of other angiogenic factors have also been shown to be involved in this process (120). It has been suggested that angiogenic factors secreted by the embryo signal to the endometrium to prepare for implantation (173). Embryo secretion of HRG by might be involved in this intricate communication. Also, those homozygous for the minor allele, HRG 633T have an increased risk for recurrent miscarriage (123). Additionally, HRG is associated with early-onset preeclampsia (125, 126), which is thought to be caused by inadequate placentation.

44

Proposed mechanisms of HRG function in female reproduction

HRG, molecular structure and plasma levels The HRG C633T polymorphism has not been detected in other species than humans. The HRG minor allele (HRG 633T) in humans is the predominant HRG genotype in other species and allows for a glycosylation in the protein (90, 92, 174). This glycosylation is located near a disulphide bond between the N2 domain and the proteolytic HRR/PRR fragment (Cys 185 – Cys 407) of the HRG protein (Figure 3). Thus, it might be speculated that the glycosylation directly affects the stability of the disulphide bond (174) and could potentially affect the release of the HRR/PRR fragment. Considering that this fragment is associated with HRG’s anti-angiogenic function (101, 175), the HRG C633T genotype might influence how HRG regulates angiogenesis in fertility.

Other mechanisms how HRG affects pregnancy and IVF parameters include variations in plasma levels, degradation and/or three dimensional structure differences. HRG levels are positively correlated to age (81) and negatively correlated with estrogen levels (121). Differences in glycosylation could possibly affect protein stability and/or degradation rates and thereby affect concentration. A recent study indicates that HRG may be phosphorylated (93). Age-related variations of plasma proteins levels have been proposed to be correlated to phosphorylation levels (176, 177). It would be interesting to study if HRG genotype also exhibits differences in phosphorylation and if this has an impact on protein stability, degradation or three-dimensional molecular structure. Glycosylation, phosphorylation and/or three dimension structure might affect ligand interactions.

HRG and ligand binding affecting implantation and placentation A proposed mechanism of how HRG affects implantation might be through building a complex with plasminogen and/or TSP. This would aid ECM degradation and angiogenesis during implantation/placentation (Figure 5). The glycosylation of the HRG molecule might alter the formation of this complex. The HRG secreted by the blastocyst could locally bind to cell surface-bound HS on the endometrium (178) and then tether plasminogen (99) at the site of implantation. This would make plasminogen conversion to plasmin more efficient, thereby increasing ECM degradation and allowing for cell migration. Additionally, blastocysts express urokinase-type plasminogen activator (179) and have increased surface tissue plasminogen activator (180), both of which increase conversion to plasmin. Mouse embryos with reduced levels of plasminogen activator do not implant (181).

Additionally, TSP, plasminogen and HRG can form a tri-molecular functioning complex (182). TSP’s anti-angiogenic activity is mediated via a structural domain known as the TSP type I repeat that binds to the CD36

45

receptor. The region where the HRG C633T SNP is located is homologous to the region in CD36 and binds to TSP with high affinity. This modulates the anti-angiogenic effect mediated by TSP/CD36 (97). The interaction between HRG and TSP/CD36 might regulate both hypercoagulability and angiogenic balance, processes that need to be well regulated for proper implantation and placentation.

Figure 5. Proposed mechanism of HRG and implantation. HS = heparan sulfate; uPA = urokinase-type plasminogen activator; tPA = tissue plasminogen activator; ECM = extracellular matrix; TSP = thrombospondin. Dashed lines represent newly formed blood vessels. The blastocyst (oval with shaded area) secretes HRG. The HRG/plasminogen complex plus tPA and uPA on or secreted by the blastocyst increases conversion to plasmin, thereby, increasing degradation of the ECM. TSP also interacts with the HRG complex increasing angiogenesis at the site of implantation.

SHB and female fertility This is the first study of SHB and human reproduction. The main focus was on three different SNPs in SHB (rs2025439, rs13298451 and rs7873102). To our knowledge, there is only one previous study on any of these SNPs, which was a genome wide study finding a link between rs7873102 and brain structure abnormalities in Alzheimer patients (183). No differences were found in the prevalence of these SNPs between normally fertile and infertile women. BMI, gonadotropin dosages administered, the percentage of immature oocytes, the number of fertilized oocytes, the percentage of good-quality embryos and the day of embryo transfer in IVF seem to be associated with SHB genotype.

The rs2025439 SNP is located in an intron in a different gene, the SLC25A51 gene. This gene is a member of the mitochondrial carrier family,

46

which consists of a vast array of proteins involved in cell metabolism and intercellular transportation (184). It may be that this intron regulates SHB gene expression. Approximately nine percent of genes overlap and regulato-ry elements of gene expression can be located in the introns of adjacent genes (185).

The 111 haplotype was predicted to be the most common. Due to the lim-ited size of the population, it was not possible to compare haplotype groups so the effect of genotype within each SNP was investigated separately. It was hypothesized that being homozygous for the minor allele (2/2) would be least advantageous for all SNPs studied. There were tendencies supporting this hypothesis as this group had fewer fertilized oocytes, a lower percent of good-quality embryos and none underwent ET day 4 – 5.

BMI differed between genotype groups both for the IVF group (rs7873102) and also for the control group (rs13298451). This is the first study linking BMI in humans to SHB. Over-expression of SHB in beta cells of transgenic mice has been associated with improved glucose homeostasis. However, the beta cells respond to cytokines with increased cell-death (186, 187). Shb knockout mice have impaired glucose homeostasis with elevated basal glucose concentrations (146) and impaired insulin secretion. Also, knockout mice have a higher birth weight (144).

Ovaries and oocytes There were no differences related to SHB genotype and levels of AMH, bFSH or AFC. Although this study has not investigated the actual number of follicles remaining in the ovaries, these markers are indirectly associated with the pool of oocytes from primary follicles to mature follicles (188). Thus, the genotype did not seem to affect the pool of oocytes at a stage that would be sensitive to gonadotropins. In contrast, a study of Shb knockout mice found that (144) wild-type mice had more follicles at these stage com-pared with knockout mice. However, knockout studies only examine differ-ences in whether the existence of the protein affects an outcome whereas in this thesis, all participants produce SHB but differences might be due to concentration levels and/or function.

The gonadotropin dosages given and the number of fertilized oocytes dif-fered between SHB genotypes. This could be due to an altered maturation of the oocyte/follicles as a difference in the percentage of immature oocytes also was found. Because not all follicles contain oocytes, it would have been interesting to investigate if the number of mature follicles differed between genotype groups. Separation of the cumulus oorphorous from the follicle is part of the maturation process. It has been suggested that the cumulus oor-phorous are not released in women with empty follicle syndrome (189). Similarity between the number of mature follicles and oocytes obtained is furthermore associated with pregnancy outcome (190).

47

Comparable to the findings in this thesis that SHB genotype affects the percentage of immature oocytes, Shb knockout mice have retarded follicle maturation and a wider time span of the initiation of meiosis I. Additionally, they have an increased number of immature oocytes (144). Contrastingly, a study of gene expression in cumulus cells from MI compared with MII oo-cytes (161) did not find a difference in SHB. However, they did not include germinal vesicle (GV) oocytes as in this thesis. Gene expression in cumulus cells might not reflect differences in gene expression throughout the whole maturation cycle. Furthermore, the effect of the SHB SNPs on oocyte maturi-ty may not be through changes in gene expression. Though it is not known how these SHB introns function, introns previously have been shown to af-fect transcription initiation or termination locations, serve as scaffolding to assist assembly of nucleosomes, be involved in alternative splicing, affect post-transcription modulations such as protein methylation, recruitment of proteins to mRNA, and affect translation yield (191).

The fertilization capacity of the oocytes did not differ between genotypes. A study on blastocyst development and IVF in Shb knockout mice also did not report any difference in fertilization rates (144).

Well-regulated angiogenesis is essential in the ovary (192, 193) for de-veloping follicles and oocytes to receive nutrients, oxygen and hormones and for removal of metabolites. Altered vasculature has been found in Shb knockout mice (194), although it is not known if this also occurs in the ova-ry. SHB operates downstream of receptors involved in angiogenesis such as VEGF-2 receptors and FGF-receptor-1 (132, 133). Variations in isoforms of VEGF or FGF have been associated with folliculogenesis and oocyte devel-opment (151, 195).

There are several plausible signaling pathways through which SHB could affect oocyte maturation. SHB signaling is associated with elevated basal extracellular regulated kinase (ERK) activity in oocytes from Shb knockout mice (144); with elevated basal focal adhesion kinase (FAK) and Rac1 (135, 196) in endothelial cells; and with FAK and Rac1 in hematopoietic stem/progenitor cells (197). Most studies indicate FAK as being essential for SHB signaling (128). Oocyte expression of FAK is elevated and may affect oocyte maturation, apoptosis and fertilization (198). SHB also provides a link between Src-family kinases and FAK (128). Src-family kinases are vital for pronuclear congression (199), resumption of meiosis (200, 201) and for the first mitotic division (202, 203). Additionally, ERK signaling is initiated by the epidermal growth factor receptor activation. These receptors are found on all ovarian (18) cell types. Gonadotropins regulate the epidermal growth factor receptor in a normal menstrual cycle (18). Epidermal growth factors are produced by the ovary and are involved in steroidogenesis, induc-tion of gonadotropin receptors, maturation of the oocyte and stimulation of other growth factors. (18).

48

A different pathway could be through insulin growth factor-1 (IGF-1). IGF-1 signals via IRS and SHP2 (18), both of which interact with SHB (128). Although it is not known if SHB is involved in this pathway in the ovary, IGF-1 is associated with granulosa cell survival (204). Together with LH, it induces androgen production (205).

Embryo development The number of good-quality embryos differed depending on genotype. Addi-tionally, the group with the minor allele underwent ET exclusively with cleavage stage embryos and had few or no embryos cryopreserved day 4 - 6. Contrastingly, opposite tendencies were seen for those with the major allele. This might be due to the difference in the number of fertilized oocytes. However, because it is known that Shb affects oocyte maturity (144), an alternative explanation might be found in differences in oocyte quality or embryo development. Shb knockout mice have impaired early embryo de-velopment both from IVF and natural conception (144). A loss of Shb -/- embryos has been demonstrated and is thought to be caused by impaired early embryo development either at implantation or through resorption (143).

49

Conclusions

HRG is present and distributed in most cells of the human female reproductive tract and placenta. No gene expression was detected indicating that HRG in these tissues is derived from the liver and transported in plasma.

HRG is present in the cytoplasm of all cells in the embryo. The distribution of HRG within the nuclei of the embryo’s cells varies depending on developmental stage. Blastocysts express and secrete HRG. The HRG C633T SNP seems to be associated with pregnancy results in IVF. The HRG C633T SNP is associated with the dosages of gonadotropin given, the number of oocytes retrieved and the number fertilized during IVF. Additionally, there may be an increased risk of only developing embryos unfit for transfer in the HRG 633T SNP group. Thus, this SNP seems to be important for ovarian response. SNPs in the SHB gene appear to be associated with prognostic markers linked with the chance of pregnancy for women undergoing IVF. BMI, gonadotropin dosages, the percentage of immature oocytes, the number of fertilized oocytes, the percentage of good-quality embryos and the day of embryo transfer in IVF seem to be influenced by genotype. The SNP prevalences did not differ between the infertile and control group. Thus, the SHB SNPs studied seem to be important for oocyte maturity and embryo quality. In conclusion, HRG and SHB appear to affect human female fertility. The HRG C633T SNP seems to influence the sensitivity of the ovaries to gonadotropins and the development of the embryo in IVF. It may also be involved in the communication between the endometrium and blastocyst at implantation and, hence, the chance of pregnancy. The SHB SNPS studied appear to affect ovarian sensitivity, oocyte maturation and embryo development in IVF. The HRG C633T SNP and the studied SNPs of the SHB gene are potential biomarkers of fertility that might be used in the future for predicting the chance of pregnancy in infertile couples.

50

Clinical Implications, at present and in the future

Infertility is a major problem for numerous couples worldwide. Many couples never receive an answer to why they are infertile and some remain childless, even after treatment. Further knowledge of reproductive mechanisms and potential biomarkers is crucial for understanding and treating infertility.

Prior knowledge of a patient’s HRG or SHB genotypes may increase diagnostic and pregnancy prediction precision and, hence, improve counseling of patients. Some may be advised to wait, and others to start treatment earlier or even consider alternatives, such as oocyte donation or adoption. Individualization of treatment has been shown to be beneficial (206-208) and knowledge of genotype might improve dosage accuracy and treatment protocols. This could optimize pregnancy chance while reducing risks, such as ovarian hyper-stimulation syndrome or cancelled treatments. Additionally, pharmacogenetics may allow for genotype-based medications/therapy.

Within the laboratory, knowledge of genotype could give embryos with higher risks priority treatment. Because SHB genotype can influence the number of immature oocytes, usage of ICSI may be more restrictive or timing delayed to allow for any possible post-retrieval maturation. HRG proteomic profiling of microfluidics might be used to improve embryo selection (173, 209) or affect strategies regarding length of embryo culture. Perhaps HRG could be a potential medium supplement if it was found to increase embryo quality or pregnancy results. Also, investigating the interaction between the mother’s and embryo’s genotype on implantation may elucidate to what extent this affects implantation.

Within female fertility, larger studies of HRG and SHB are needed, especially studies that involve miscarriage, clinical pregnancy and live birth. Comparing genotype frequencies within sub-groups of patients such as low- or high responders may explain possible genotype effects. The correlation between time to pregnancy and genotype could be studied in fertile couples or by comparing frequencies in couples with recurrent IVF failure. Additionally, studying the effect of SHB on oocytes cultured for in vitro maturation may give further information on the maturation process.

51

Fertility is also dependent on the male and HRG and SHB have not been studied regarding male fertility. Further studies are warranted to determine if sperm quality is affected. Sperm is stored in the cervix and Fallopian tubes (30) and studying the effect of the different female genotypes on the fertilization capacity of sperm may further knowledge of reproduction. Also, there is a need to investigate the combined genotype of the couple and chance of pregnancy.

It would be interesting to build and evaluate a computer model containing HRG and SHB together with other known biomarkers. Using such a model, treatment protocols, lab routines or embryo assessment could be improved, and thus improve the chance of pregnancy for patients.

52

Summary in Swedish – Sammanfattning på svenska

Enligt Världshälsorganisationen (WHO) är definitionen på ofrivillig barn-löshet när ett par i fertil ålder inte blivit gravida efter att aktivt ha försökt under minst ett år (Zegers-Hochschild et al., 2009). Omkring 15 % av alla par i fertil ålder drabbas av ofrivillig barnlöshet, vilket motsvarar cirka 60 - 80 miljoner par globalt. Orsaken till varför vissa par inte blir gravida spon-tant är oklar hos cirka 22 % av de som utreds. Ett sätt att hjälpa dessa par kan vara via assisterad befruktning s.k. in vitro fertilisering (IVF) men drygt 30 % av paren får inte barn efter tre IVF försök.

Många olika biologiska processer som t.ex. blodkärlsnybildning, immu-nologiska processer och programmerad celldöd är viktiga i regleringen av kvinnlig fertilitet. Mikromiljön runt ägget (oocyten), embryot och fostret är avgörande för att en normal graviditet ska kunna utvecklas. För att uppnå ökad kunskap om reproduktion samt för att hitta potentiella framtida dia-gnostiska verktyg och behandlingsmetoder för infertila par har i denna av-handling skillnader i gen- och proteinuttryck kopplats till medicinska variab-ler och studerats och jämförts mellan infertila och fertila grupper av kvinnor.

HRG är ett plasmaprotein som reglerar ett flertal biologiska system, t.ex. angiogenes, immunsystemet och koagulation/fibrinolys genom att bilda komplex med olika ligander. I det första delarbetet studerades HRGs före-komst och genuttryck i kvinnans inre genitalia samt i embryon. Med Wes-tern blot detekterades HRG i follikelvätska och med immunhistokemiska analyser sågs förekomst i äggledarna, endometriet, myometriet och placenta. HRG detekterades också i varierande omfattning i embryots cellkärna bero-ende på utvecklingsstadium. Genuttryck av HRG har tidigare hittats i levern men i denna studie sågs även att det tidiga embryot uttrycker HRG mRNA. Vidare analys av mediet som embryot odlats i visade dessutom att HRG utsöndrats till mediet.

I det andra delarbetet studerades hur en specifik genetisk variation, en s.k. ”single nucletide polymorphism” (SNP), i HRG kallad HRG C633T påverkar graviditetsresultatet vid IVF. Follikelvätska från 24 kvinnor med olika infer-tilitetsorsaker analyserades med Western blot och den specifika HRG-polymorfin kunde detekteras på protein-nivå. En koppling hittades mellan HRG C633T genotyp och graviditetsutfall. Homozygota bärare av HRG C633 hade högst graviditetsfrekvens, de heterozygota bärarna intermediär

53

frekvens och bland de kvinnor som var homozygota för HRG 633T blev i detta material inte någon gravid. Ytterligare en grupp kvinnor (n = 34) in-kluderades i syfte att verifiera resultaten. I denna grupp kvinnor hade alla diagnosen oförklarad infertilitet. Vid genotypning för HRG C633T SNP bekräftades resultaten, d.v.s. att HRG C633 är kopplad till en betydligt högre graviditetsfrekvens vid IVF än HRG 633T.

I den uppföljande studien (delarbete tre) rekryterades en större grupp kvinnor (n = 155) med varierande infertilitetsorsak. En sammanställning över medicinska bakgrundsfaktorer, behandling, IVF-parametrar samt gravi-ditetsutfall gjordes. Alla kvinnor genotypades för HRG C633T och vid en subgrupp-analys av kvinnorna med oförklarad infertilitet (n = 67) noterades att de som var homozygota för HRG 633T utvecklade färre oocyter trots högre gonadotropindoser under IVF-behandlingen. I samma grupp förelåg också större risk för att embryot skulle vara av så låg kvalitet att något em-bryo inte skulle kunna återföras till livmodern.

Src homology 2 domain-containing adapter protein B (SHB) är ett intra-cellulärt adaptorprotein som bildar signaleringskomplex vid receptor aktive-ring. SHB reglerar angiogenes, immunsystemet, cellproliferation och apop-tos. Shb knockout möss har ändrad oocyt/follikel mognadsprocess och för-sämrad embryo- och fosterutveckling. SHBs betydelse för human reprodukt-ion har inte studerats tidigare. I det fjärde delarbetet användes DNA från samma studiepopulation som i delarbete tre men en kontrollgrupp bestående av normalt fertila kvinnor inkluderades också. Följande SHB SNP:s analyse-rades: rs2025439 (n = 140 IVF; n = 97 kontroller), rs13298451 (n = 140 IVF; n = 98 kontroller) and rs7873102 (n = 139 IVF; n = 98 kontroller). De olika genotyperna var lika vanliga bland infertila som i kontrollgruppen. Däremot skilde sig BMI, gonadotropindoser, andel omogna oocyter, antal befruktade oocyter, andelen embryon av hög kvalitet embryo/oocyt vid da-gen för embryoåterföring åt mellan genotyperna.

Sammanfattningsvis visar delstudierna att HRG och SHB är två proteiner av betydelse för kvinnlig fertilitet. Resultaten kan förhoppningsvis bidra till en ökad förståelse av de centrala mekanismer som reglerar reproduktion. Fortsatta studier inom området skulle kunna möjliggöra utvecklingen av prediktionsmodeller för sannolikheten att lyckas vid assisterad befruktning samt öka möjligheterna till optimering av behandling vid infertilitet, vilket skulle kunna bli till stor nytta och glädje för infertila par. Kännedom om parets genotyp skulle kunna användas för att optimera rådgivning, behand-lingsprotokoll och resultat.

54

Acknowledgements

This thesis work was conducted at the Department of Women´s and Children´s Health, Uppsala University, Sweden. I would like to express my sincerest gratitude to all participating patients and to all who supported and assisted me especially: Helena Åkerud, Professor, Department of Women´s and Children’s Health, main supervisor. You are an amazingly productive, brilliant, focused, motivated, ambitious, dedicated role model! Thank you for taking me on as a PhD student and for your continual belief in my capabilities. Your support and understanding has been wonderful. You have taught me so much about so many different aspects of research. Karin Kårehed, MD, PhD, co-supervisor. Your in-depth of knowledge about biology has enriched many enjoyable discussions on such varied aspects and levels. Thanks for supportive pep-talks! You have an amazing ability to spread calmness when imminent dangers such as scary deadlines arise. Agneta Skoog Svanberg, Associate professor, Department of Women´s and Children’s Health, co-supervisor. Thank you for your generosity! It has been a true pleasure and honor working with you. Thank you for allowing me (almost) full reins to do our project from the position I wanted to. What fun it was to get our article accepted so quickly. Anneli Stavreus-Evers, Associate professor, Department of Women´s and Children’s Health, co-supervisor. I always enjoyed our chats when I arrived with my samples for the freezer! Thanks for ethical discussions and for sharing your knowledge about the biomolecular aspects of implantation. Jan Gustafsson, Professor, former Department Head and Agneta Skoog Svanberg, Associate Professor, presiding Department Head and Children’s Health, Uppsala University, for the opportunity to carry out my doctoral studies. Coauthors: Fredwell Hambiliki, for exciting discoveries at the microscope and kind support. Kjell Wånggren, for helpful suggestions. Judith

55

Menezes, for sharing your knowledge and love of embryos and the laborato-ry. Torbjörn Bergh, Jan Holte, Oddvar Bakos and Thomas Brodin, col-leagues at Carl von Linné Clinic. Thank you for providing a work environ-ment that encourages and supports, in every way possible, research. It’s in-spirational to work with such fantastic clinicians with amazing research backgrounds. Thanks for all the support and wise guidance. Håkan Wramsby and Carolina Tristen, former Branch Executive Direc-tors at Fertilitetscentrum Stockholm for making it possible to carry out re-search even during periods when short on staff. Thanks for practical assis-tance enabling me to get through some necessary formal requirements. Former co-workers at Fertilitetscentrum for help with recruiting patients and samples. Special thanks to: Lalit Kumar PGL for discussions about research and implantation. Katarina Vaegter for supportive discussions on how to juggle a family, career and research. Kristina Hjalmarsson for all extra help ensuring we did not miss any samples from recruited patients.

Professors Matts Olovsson, Bengt Sorbe, Gunilla Sydsjö and Associate professor Helle Kieler for wise guidance, inspiration and exciting discus-sions. Professor Michael Welsh for introducing me to the exciting world of SHB. Evangelia Elenis for a fruitful cooperation and interesting discussions. Malin Olsson, Karin Lindgren, Christine Sund Lundström and Sylvia Malmberg for technical assistance in the laboratory. Personnel at RPC, Uppsala University Hospital, and Reproductive Med-icine, Huddinge Hospital, for patient recruitment and collection of samples. The Carl von Linné Clinic team for smiles, friendship and great times. My friends for unconditional friendship, fun distractions and support. My father whose wise advice along the way has been invaluable and for repeatedly showing me what is important in life. My children, Jacob, Miranda, Amelie and Matilda, who continually show me the miracle of parenthood. My beloved Rune for infinite support in every way imaginable.

56

57

References

1. Clarke, G.N. 2006. A.R.T. and history, 1678-1978. Hum Reprod 21:1645-1650.

2. Tata, J. 2005. One hundred years of hormones. EMBO report 6:490-496.

3. Pincus, G., and Enzmann, E.V. 1935. The comparative behavior of mammalian eggs in vivo and in vitro: I. The activation of ovarian eggs. J Exp Med 62:665-675.

4. Rock, J., and Menkin, M.F. 1944. In vitro fertilization and cleavage of human ovarian eggs Science 100:105-107.

5. Bavister, B.D. 2002. Early history of in vitro fertilization. Reproduction 124:181-196.

6. Gemzell, C., and Kjessler, B. 1964. Treatment of infertility after partial hypophysectomy with human pituitary gonadotropins. Lancet 1:644.

7. Steptoe, P.C., and Edwards, R.G. 1976. Reimplantation of a human embryo with subsequent tubal pregnancy. Lancet 1:880-882.

8. Steptoe, P.C., and Edwards, R.G. 1978. Birth after the reimplantation of a human embryo. Lancet 2:366.

9. Zeilmaker, G.H., Alberda, A.T., van Gent, I., Rijkmans, C.M., and Drogendijk, A.C. 1984. Two pregnancies following transfer of intact frozen-thawed embryos. Fertil Steril 42:293-296.

10. Handyside, A.H., Pattinson, J.K., Penketh, R.J., Delhanty, J.D., Winston, R.M., and Tuddenham, E.G. 1989. Biopsy of human preimplantation embryos and sexing by DNA amplification. Lancet 1:347-349.

11. Palermo, G., Joris, H., Devroey, P., and Van Steirteghem, A.C. 1992. Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet 340:17-18.

12. Hillier, S.G. 2013. IVF endocrinology: the Edwards era. Mol Hum Reprod 19:799-808.

13. Dorland, W.A.N. 2012. Dorland's illustrated medical dictionary. Philadelphia, Pa: Elsevier/Saunders.

14. Crosignani, P.G., and Rubin, B.L. 2000. Optimal use of infertility diagnostic tests and treatments. The ESHRE Capri Workshop Group. Hum Reprod 15:723-732.

15. te Velde, E.R., Eijkemans, R., and Habbema, H.D. 2000. Variation in couple fecundity and time to pregnancy, an essential concept in human reproduction. Lancet 355:1928-1929.

58

16. Zegers-Hochschild, F., Adamson, G.D., de Mouzon, J., Ishihara, O., Mansour, R., Nygren, K., Sullivan, E., and Vanderpoel, S. 2009. International Committee for Monitoring Assisted Reproductive Technology (ICMART) and the World Health Organization (WHO) revised glossary of ART terminology, 2009. Fertil Steril 92:1520-1524.

17. Haller-Kikkatalo, K., Salumets, A., and Uibo, R. 2012. Review on autoimmune reactions in female infertility: antibodies to follicle stimulating hormone. Clin Dev Immunol 2012:762541.

18. Fauser, B.C.J.M. 2003. Reproductive medicine: molecular, cellular, and genetic fundamentals. Boca Raton: Parthenon Pub. Group. 296-308; 367-378 pp.

19. Feng, C.W., Bowles, J., and Koopman, P. 2014. Control of mammalian germ cell entry into meiosis. Mol Cell Endocrinol 382:488-497.

20. Kerr, J.B., Myers, M., and Anderson, R.A. 2013. The dynamics of the primordial follicle reserve. Reproduction 146:R205-215.

21. Da Silva-Buttkus, P., Marcelli, G., Franks, S., Stark, J., and Hardy, K. 2009. Inferring biological mechanisms from spatial analysis: prediction of a local inhibitor in the ovary. Proc Natl Acad Sci U S A 106:456-461.

22. Sutton, M.L., Gilchrist, R.B., and Thompson, J.G. 2003. Effects of in-vivo and in-vitro environments on the metabolism of the cumulus-oocyte complex and its influence on oocyte developmental capacity. Hum Reprod Update 9:35-48.

23. Ackert, C.L., Gittens, J.E., O'Brien, M.J., Eppig, J.J., and Kidder, G.M. 2001. Intercellular communication via connexin43 gap junctions is required for ovarian folliculogenesis in the mouse. Dev Biol 233:258-270.

24. Matzuk, M.M., Burns, K.H., Viveiros, M.M., and Eppig, J.J. 2002. Intercellular communication in the mammalian ovary: oocytes carry the conversation. Science 296:2178-2180.

25. Ben-Ami, I., Freimann, S., Armon, L., Dantes, A., Strassburger, D., Friedler, S., Raziel, A., Seger, R., Ron-El, R., and Amsterdam, A. 2006. PGE2 up-regulates EGF-like growth factor biosynthesis in human granulosa cells: new insights into the coordination between PGE2 and LH in ovulation. Mol Hum Reprod 12:593-599.

26. Lyons, R.A., Saridogan, E., and Djahanbakhch, O. 2006. The effect of ovarian follicular fluid and peritoneal fluid on Fallopian tube ciliary beat frequency. Hum Reprod 21:52-56.

27. Lee, K.F., and Yeung, W.S. 2006. Gamete/embryo - oviduct interactions: implications on in vitro culture. Hum Fertil (Camb) 9:137-143.

28. Sun, F., Giojalas, L.C., Rovasio, R.A., Tur-Kaspa, I., Sanchez, R., and Eisenbach, M. 2003. Lack of species-specificity in mammalian sperm chemotaxis. Dev Biol 255:423-427.

59

29. Yao, Y., Ho, P., and Yeung, W.S. 1999. Effects of human follicular fluid on spermatozoa that have been cocultured with human oviductal cells. Fertil Steril 72:1079-1084.

30. Hunter, R.H. 1995. Human sperm reservoirs and Fallopian tube function: a role for the intra-mural portion? Acta Obstet Gynecol Scand 74:677-681.

31. Braude, P., Bolton, V., and Moore, S. 1988. Human gene expression first occurs between the four- and eight-cell stages of preimplantation development. Nature 332:459-461.

32. Lee, D.R., Lee, J.E., Yoon, H.S., Lee, H.J., Kim, M.K., and Roh, S.I. 1997. The supplementation of culture medium with protease improves the hatching rate of mouse embryos. Hum Reprod 12:2493-2498.

33. Van Sinderen, M., Menkhorst, E., Winship, A., Cuman, C., and Dimitriadis, E. 2013. Preimplantation human blastocyst-endometrial interactions: the role of inflammatory mediators. Am J Reprod Immunol 69:427-440.

34. Dey, S.K., Lim, H., Das, S.K., Reese, J., Paria, B.C., Daikoku, T., and Wang, H. 2004. Molecular cues to implantation. Endocr Rev 25:341-373.

35. Brandes, M., Hamilton, C.J., de Bruin, J.P., Nelen, W.L., and Kremer, J.A. 2010. The relative contribution of IVF to the total ongoing pregnancy rate in a subfertile cohort. Hum Reprod 25:118-126.

36. Unuane, D., Tournaye, H., Velkeniers, B., and Poppe, K. 2011. Endocrine disorders & female infertility. Best Pract Res Clin Endocrinol Metab 25:861-873.

37. Hull, M.G., Glazener, C.M., Kelly, N.J., Conway, D.I., Foster, P.A., Hinton, R.A., Coulson, C., Lambert, P.A., Watt, E.M., and Desai, K.M. 1985. Population study of causes, treatment, and outcome of infertility. Br Med J (Clin Res Ed) 291:1693-1697.

38. Metwally, M., Farquhar, C.M., and Li, T.C. 2011. Is another meta-analysis on the effects of intramural fibroids on reproductive outcomes needed? Reprod Biomed Online 23:2-14.

39. Holoch, K.J., and Lessey, B.A. 2010. Endometriosis and infertility. Clin Obstet Gynecol 53:429-438.

40. Ozkan, S., Murk, W., and Arici, A. 2008. Endometriosis and infertility: epidemiology and evidence-based treatments. Ann N Y Acad Sci 1127:92-100.

41. Tocci, A., and Lucchini, C. 2010. WHO reference values for human semen. Hum Reprod Update 16:559; author reply 559.

42. Esteves, S.C., Hamada, A., Kondray, V., Pitchika, A., and Agarwal, A. 2012. What every gynecologist should know about male infertility: an update. Arch Gynecol Obstet.

43. Rantala, M.L., and Koskimies, A.I. 1988. Sexual behavior of infertile couples. Int J Fertil 33:26-30.

60

44. Gleicher, N., and Barad, D. 2006. Unexplained infertility: does it really exist? Hum Reprod 21:1951-1955.

45. Brandes, M., Hamilton, C.J., van der Steen, J.O., de Bruin, J.P., Bots, R.S., Nelen, W.L., and Kremer, J.A. 2011. Unexplained infertility: overall ongoing pregnancy rate and mode of conception. Hum Reprod 26:360-368.

46. Svensk Förening för Obstetrik och Gynekologi, A.R.G. 2010. Ofrivillig barnlöshet.

47. Broekmans, F.J., Soules, M.R., and Fauser, B.C. 2009. Ovarian aging: mechanisms and clinical consequences. Endocr Rev 30:465-493.

48. Nelson, S.M., Telfer, E.E., and Anderson, R.A. 2013. The ageing ovary and uterus: new biological insights. Hum Reprod Update 19:67-83.

49. Broekmans, F.J., Kwee, J., Hendriks, D.J., Mol, B.W., and Lambalk, C.B. 2006. A systematic review of tests predicting ovarian reserve and IVF outcome. Hum Reprod Update 12:685-718.

50. La Marca, A., and Sunkara, S.K. 2014. Individualization of controlled ovarian stimulation in IVF using ovarian reserve markers: from theory to practice. Hum Reprod Update 20:124-140.

51. Nelson, S.M. 2013. Biomarkers of ovarian response: current and future applications. Fertil Steril 99:963-969.

52. Wang, Q., and Sun, Q.Y. 2007. Evaluation of oocyte quality: morphological, cellular and molecular predictors. Reprod Fertil Dev 19:1-12.

53. Alpha-Scientists-in-Reproductive-Medicine, and ESHRE-Special-Interest-Group-of-Embryology. 2011. The Istanbul consensus workshop on embryo assessment: proceedings of an expert meeting. Hum Reprod 26:1270-1283.

54. Adamson, G.D., and Baker, V.L. 2003. Subfertility: causes, treatment and outcome. Best Pract Res Clin Obstet Gynaecol 17:169-185.

55. Olivius, K., Friden, B., Lundin, K., and Bergh, C. 2002. Cumulative probability of live birth after three in vitro fertilization/intracytoplasmic sperm injection cycles. Fertil Steril 77:505-510.

56. Leushuis, E., van der Steeg, J.W., Steures, P., Bossuyt, P.M., Eijkemans, M.J., van der Veen, F., Mol, B.W., and Hompes, P.G. 2009. Prediction models in reproductive medicine: a critical appraisal. Hum Reprod Update 15:537-552.

57. Waylen, A.L., Metwally, M., Jones, G.L., Wilkinson, A.J., and Ledger, W.L. 2009. Effects of cigarette smoking upon clinical outcomes of assisted reproduction: a meta-analysis. Hum Reprod Update 15:31-44.

58. Glujovsky, D., Blake, D., Farquhar, C., and Bardach, A. 2012. Cleavage stage versus blastocyst stage embryo transfer in assisted reproductive technology. Cochrane Database Syst Rev 7:CD002118.

61

59. van Loendersloot, L.L., van Wely, M., Limpens, J., Bossuyt, P.M., Repping, S., and van der Veen, F. 2010. Predictive factors in in vitro fertilization (IVF): a systematic review and meta-analysis. Hum Reprod Update 16:577-589.

60. Brodin, T., Bergh, T., Berglund, L., Hadziosmanovic, N., and Holte, J. 2008. Menstrual cycle length is an age-independent marker of female fertility: results from 6271 treatment cycles of in vitro fertilization. Fertil Steril 90:1656-1661.

61. Brodin, T., Bergh, T., Berglund, L., Hadziosmanovic, N., and Holte, J. 2009. High basal LH levels in combination with low basal FSH levels are associated with high success rates at assisted reproduction. Hum Reprod 24:2755-2759.

62. Brodin, T., Hadziosmanovic, N., Berglund, L., Olovsson, M., and Holte, J. 2013. Antimullerian hormone levels are strongly associated with live-birth rates after assisted reproduction. J Clin Endocrinol Metab 98:1107-1114.

63. Holte, J., Brodin, T., Berglund, L., Hadziosmanovic, N., Olovsson, M., and Bergh, T. 2011. Antral follicle counts are strongly associated with live-birth rates after assisted reproduction, with superior treatment outcome in women with polycystic ovaries. Fertil Steril 96:594-599.

64. The-ESHRE-Capri-Workshop-Group. 2010. Europe the continent with the lowest fertility. Hum Reprod Update 16:590-602.

65. Watson, J.D., and Crick, F.H.C. 1954. The structure of DNA. Spring Harb. Symp. Quant. Biol 18:123–131.

66. Perez Mayorga, M., Gromoll, J., Behre, H.M., Gassner, C., Nieschlag, E., and Simoni, M. 2000. Ovarian response to follicle-stimulating hormone (FSH) stimulation depends on the FSH receptor genotype. J Clin Endocrinol Metab 85:3365-3369.

67. Moron, F.J., and Ruiz, A. 2010. Pharmacogenetics of controlled ovarian hyperstimulation: time to corroborate the clinical utility of FSH receptor genetic markers. Pharmacogenomics 11:1613-1618.

68. Daelemans, C., Smits, G., de Maertelaer, V., Costagliola, S., Englert, Y., Vassart, G., and Delbaere, A. 2004. Prediction of severity of symptoms in iatrogenic ovarian hyperstimulation syndrome by follicle-stimulating hormone receptor Ser680Asn polymorphism. J Clin Endocrinol Metab 89:6310-6315.

69. Klinkert, E.R., te Velde, E.R., Weima, S., van Zandvoort, P.M., Hanssen, R.G., Nilsson, P.R., de Jong, F.H., Looman, C.W., and Broekmans, F.J. 2006. FSH receptor genotype is associated with pregnancy but not with ovarian response in IVF. Reprod Biomed Online 13:687-695.

70. Altmae, S., Haller, K., Peters, M., Hovatta, O., Stavreus-Evers, A., Karro, H., Metspalu, A., and Salumets, A. 2007. Allelic estrogen receptor 1 (ESR1) gene variants predict the outcome of ovarian stimulation in in vitro fertilization. Mol Hum Reprod 13:521-526.

62

71. Ayvaz, O.U., Ekmekci, A., Baltaci, V., Onen, H.I., and Unsal, E. 2009. Evaluation of in vitro fertilization parameters and estrogen receptor alpha gene polymorphisms for women with unexplained infertility. J Assist Reprod Genet 26:503-510.

72. Georgiou, I., Konstantelli, M., Syrrou, M., Messinis, I.E., and Lolis, D.E. 1997. Oestrogen receptor gene polymorphisms and ovarian stimulation for in-vitro fertilization. Hum Reprod 12:1430-1433.

73. Laanpere, M., Altmae, S., Kaart, T., Stavreus-Evers, A., Nilsson, T.K., and Salumets, A. 2011. Folate-metabolizing gene variants and pregnancy outcome of IVF. Reprod Biomed Online 22:603-614.

74. Christianson, M.S., and Yates, M. 2012. Scavenger receptor class B type 1 gene polymorphisms and female fertility. Curr Opin Endocrinol Diabetes Obes 19:115-120.

75. Goodman, C., Jeyendran, R.S., and Coulam, C.B. 2008. Vascular endothelial growth factor gene polymorphism and implantation failure. Reprod Biomed Online 16:720-723.

76. Casarini, L., Pignatti, E., and Simoni, M. 2011. Effects of polymorphisms in gonadotropin and gonadotropin receptor genes on reproductive function. Rev Endocr Metab Disord 12:303-321.

77. Altmae, S., Hovatta, O., Stavreus-Evers, A., and Salumets, A. 2011. Genetic predictors of controlled ovarian hyperstimulation: where do we stand today? Hum Reprod Update 17:813-828.

78. Koide, T., Foster, D., Yoshitake, S., and Davie, E.W. 1986. Amino acid sequence of human histidine-rich glycoprotein derived from the nucleotide sequence of its cDNA. Biochemistry 25:2220-2225.

79. Lee, C., Bongcam-Rudloff, E., Sollner, C., Jahnen-Dechent, W., and Claesson-Welsh, L. 2009. Type 3 cystatins; fetuins, kininogen and histidine-rich glycoprotein. Front Biosci 14:2911-2922.

80. Jones, A.L., Hulett, M.D., and Parish, C.R. 2005. Histidine-rich glycoprotein: A novel adaptor protein in plasma that modulates the immune, vascular and coagulation systems. Immunol Cell Biol 83:106-118.

81. Corrigan, J.J., Jr., Jeter, M.A., Bruck, D., and Feinberg, W.M. 1990. Histidine-rich glycoprotein levels in children: the effect of age. Thromb Res 59:681-686.

82. Shigekiyo, T., Kanazuka, M., Azuma, H., Ohshima, T., Kusaka, K., and Saito, S. 1995. Congenital deficiency of histidine-rich glycoprotein: failure to identify abnormalities in routine laboratory assays of hemostatic function, immunologic function, and trace elements. J Lab Clin Med 125:719-723.

83. Saigo, K., Yoshida, A., Ryo, R., Yamaguchi, N., and Leung, L.L. 1990. Histidine-rich glycoprotein as a negative acute phase reactant. Am J Hematol 34:149-150.

84. Olsson, A.K., Larsson, H., Dixelius, J., Johansson, I., Lee, C., Oellig, C., Bjork, I., and Claesson-Welsh, L. 2004. A fragment of histidine-rich glycoprotein is a potent inhibitor of tumor vascularization. Cancer Res 64:599-605.

63

85. Thulin, A., Ringvall, M., Dimberg, A., Karehed, K., Vaisanen, T., Vaisanen, M.R., Hamad, O., Wang, J., Bjerkvig, R., Nilsson, B., et al. 2009. Activated platelets provide a functional microenvironment for the antiangiogenic fragment of histidine-rich glycoprotein. Mol Cancer Res 7:1792-1802.

86. Klenotic, P.A., Huang, P., Palomo, J., Kaur, B., Van Meir, E.G., Vogelbaum, M.A., Febbraio, M., Gladson, C.L., and Silverstein, R.L. 2010. Histidine-rich glycoprotein modulates the anti-angiogenic effects of vasculostatin. Am J Pathol 176:2039-2050.

87. Matboli, M., Eissa, S., and Said, H. 2014. Evaluation of histidine-rich glycoprotein tissue RNA and serum protein as novel markers for breast cancer. Med Oncol 31:897.

88. Wu, J., Xie, X., Liu, Y., He, J., Benitez, R., Buckanovich, R.J., and Lubman, D.M. 2012. Identification and confirmation of differentially expressed fucosylated glycoproteins in the serum of ovarian cancer patients using a lectin array and LC-MS/MS. J Proteome Res 11:4541-4552.

89. Wang, Y.S., Cao, R., Jin, H., Huang, Y.P., Zhang, X.Y., Cong, Q., He, Y.F., and Xu, C.J. 2011. Altered protein expression in serum from endometrial hyperplasia and carcinoma patients. J Hematol Oncol 4:15.

90. Kassaar, O., McMahon, S.A., Thompson, R., Botting, C.H., Naismith, J.H., and Stewart, A.J. 2014. Crystal structure of histidine-rich glycoprotein N2 domain reveals redox activity at an interdomain disulfide bridge: implications for angiogenic regulation. Blood 123:1948-1955.

91. Wakabayashi, S., and Koide, T. 2011. Histidine-rich glycoprotein: a possible modulator of coagulation and fibrinolysis. Semin Thromb Hemost 37:389-394.

92. Hennis, B.C., van Boheemen, P.A., Wakabayashi, S., Koide, T., Hoffmann, J.J., Kievit, P., Dooijewaard, G., Jansen, J.G., and Kluft, C. 1995. Identification and genetic analysis of a common molecular variant of histidine-rich glycoprotein with a difference of 2kD in apparent molecular weight. Thromb Haemost 74:1491-1496.

93. Patel, K.K., Poon, I.K., Talbo, G.H., Perugini, M.A., Taylor, N.L., Ralph, T.J., Hoogenraad, N.J., and Hulett, M.D. 2013. New method for purifying histidine-rich glycoprotein from human plasma redefines its functional properties. IUBMB Life 65:550-563.

94. Borza, D.B., Tatum, F.M., and Morgan, W.T. 1996. Domain structure and conformation of histidine-proline-rich glycoprotein. Biochemistry 35:1925-1934.

95. Poon, I.K., Patel, K.K., Davis, D.S., Parish, C.R., and Hulett, M.D. 2011. Histidine-rich glycoprotein: the Swiss Army knife of mammalian plasma. Blood 117:2093-2101.

96. Borza, D.B. 2005. Life without histidine-rich glycoprotein: modulation of the hemostatic balance revisited. J Thromb Haemost 3:863-864.

64

97. Silverstein, R.L., and Febbraio, M. 2007. CD36-TSP-HRGP interactions in the regulation of angiogenesis. Curr Pharm Des 13:3559-3567.

98. Simantov, R., Febbraio, M., and Silverstein, R.L. 2005. The antiangiogenic effect of thrombospondin-2 is mediated by CD36 and modulated by histidine-rich glycoprotein. Matrix Biol 24:27-34.

99. Jones, A.L., Hulett, M.D., Altin, J.G., Hogg, P., and Parish, C.R. 2004. Plasminogen is tethered with high affinity to the cell surface by the plasma protein, histidine-rich glycoprotein. J Biol Chem 279:38267-38276.

100. Guan, X., Juarez, J.C., Qi, X., Shipulina, N.V., Shaw, D.E., Morgan, W.T., McCrae, K.R., Mazar, A.P., and Donate, F. 2004. Histidine-proline rich glycoprotein (HPRG) binds and transduces anti-angiogenic signals through cell surface tropomyosin on endothelial cells. Thromb Haemost 92:403-412.

101. Lee, C., Dixelius, J., Thulin, A., Kawamura, H., Claesson-Welsh, L., and Olsson, A.K. 2006. Signal transduction in endothelial cells by the angiogenesis inhibitor histidine-rich glycoprotein targets focal adhesions. Exp Cell Res 312:2547-2556.

102. Wake, H., Mori, S., Liu, K., Takahashi, H.K., and Nishibori, M. 2009. Histidine-rich glycoprotein inhibited high mobility group box 1 in complex with heparin-induced angiogenesis in matrigel plug assay. Eur J Pharmacol 623:89-95.

103. Gorgani, N.N., Parish, C.R., Easterbrook Smith, S.B., and Altin, J.G. 1997. Histidine-rich glycoprotein binds to human IgG and C1q and inhibits the formation of insoluble immune complexes. Biochemistry 36:6653-6662.

104. Jones, A.L., Poon, I.K., Hulett, M.D., and Parish, C.R. 2005. Histidine-rich glycoprotein specifically binds to necrotic cells via its amino-terminal domain and facilitates necrotic cell phagocytosis. J Biol Chem 280:35733-35741.

105. Chang, N.S., Leu, R.W., Anderson, J.K., and Mole, J.E. 1994. Role of N-terminal domain of histidine-rich glycoprotein in modulation of macrophage Fc gamma receptor-mediated phagocytosis. Immunology 81:296-302.

106. Chang, N.S., Leu, R.W., Rummage, J.A., Anderson, J.K., and Mole, J.E. 1992. Regulation of macrophage Fc receptor expression and phagocytosis by histidine-rich glycoprotein. Immunology 77:532-538.

107. Kacprzyk, L., Rydengard, V., Morgelin, M., Davoudi, M., Pasupuleti, M., Malmsten, M., and Schmidtchen, A. 2007. Antimicrobial activity of histidine-rich peptides is dependent on acidic conditions. Biochim Biophys Acta 1768:2667-2680.

108. Shannon, O., Rydengard, V., Schmidtchen, A., Morgelin, M., Alm, P., Sorensen, O.E., and Bjorck, L. 2010. Histidine-rich glycoprotein promotes bacterial entrapment in clots and decreases mortality in a mouse model of sepsis. Blood 116:2365-2372.

65

109. Rydengard, V., Olsson, A.K., Morgelin, M., and Schmidtchen, A. 2007. Histidine-rich glycoprotein exerts antibacterial activity. FEBS J 274:377-389.

110. Rydengard, V., Shannon, O., Lundqvist, K., Kacprzyk, L., Chalupka, A., Olsson, A.K., Morgelin, M., Jahnen-Dechent, W., Malmsten, M., and Schmidtchen, A. 2008. Histidine-rich glycoprotein protects from systemic Candida infection. PLoS Pathog 4:e1000116.

111. Leung, L.L. 1986. Interaction of histidine-rich glycoprotein with fibrinogen and fibrin. J Clin Invest 77:1305-1311.

112. Tsuchida-Straeten, N., Ensslen, S., Schafer, C., Woltje, M., Denecke, B., Moser, M., Graber, S., Wakabayashi, S., Koide, T., and Jahnen-Dechent, W. 2005. Enhanced blood coagulation and fibrinolysis in mice lacking histidine-rich glycoprotein (HRG). J Thromb Haemost 3:865-872.

113. Lijnen, H.R., and Collen, D. 1989. Interaction of heparin with histidine-rich glycoprotein. In Ann N Y Acad Sci. 181-185.

114. Lijnen, H.R., Hoylaerts, M., and Collen, D. 1980. Isolation and characterization of a human plasma protein with affinity for the lysine binding sites in plasminogen. Role in the regulation of fibrinolysis and identification as histidine-rich glycoprotein. J Biol Chem 255:10214-10222.

115. Mori, S., Shinohata, R., Renbutsu, M., Takahashi, H.K., Fang, Y.I., Yamaoka, K., Okamoto, M., Yamamoto, I., and Nishibori, M. 2003. Histidine-rich glycoprotein plus zinc reverses growth inhibition of vascular smooth muscle cells by heparin. Cell Tissue Res 312:353-359.

116. Shatsky, M., Saigo, K., Burdach, S., Leung, L.L., and Levitt, L.J. 1989. Histidine-rich glycoprotein blocks T cell rosette formation and modulates both T cell activation and immunoregulation. J Biol Chem 264:8254-8259.

117. Brown, K.J., and Parish, C.R. 1994. Histidine-rich glycoprotein and platelet factor 4 mask heparan sulfate proteoglycans recognized by acidic and basic fibroblast growth factor. Biochemistry 33:13918-13927.

118. Johnson, L.D., Goubran, H.A., and Kotb, R.R. 2014. Histidine rich glycoprotein and cancer: a multi-faceted relationship. Anticancer Res 34:593-603.

119. Gorgani, N.N., Smith, B.A., Kono, D.H., and Theofilopoulos, A.N. 2002. Histidine-rich glycoprotein binds to DNA and Fc gamma RI and potentiates the ingestion of apoptotic cells by macrophages. J Immunol 169:4745-4751.

120. Wulff, C., Weigand, M., Kreienberg, R., and Fraser, H.M. 2003. Angiogenesis during primate placentation in health and disease. Reproduction 126:569-577.

66

121. Hennis, B.C., Boomsma, D.I., Fijnvandraat, K., Gevers Leuven, J.A., Peters, M., and Kluft, C. 1995. Estrogens reduce plasma histidine-rich glycoprotein (HRG) levels in a dose-dependent way. Thromb Haemost 73:484-487.

122. Haukkamaa, M., Morgan, W.T., and Koskelo, P. 1983. Serum histidine-rich glycoprotein during pregnancy and hormone treatment. Scand J Clin Lab Invest 43:591-595.

123. Lindgren, K.E., Karehed, K., Karypidis, H., Hosseini, F., Bremme, K., Landgren, B.M., Skjoldebrand-Sparre, L., Stavreus-Evers, A., Sundstrom-Poromaa, I., and Akerud, H. 2013. Histidine-rich glycoprotein gene polymorphism in patients with recurrent miscarriage. Acta Obstet Gynecol Scand 92:974-977.

124. Elenis, E., Lindgren, K.E., Karypidis, H., Skalkidou, A., Hosseini, F., Bremme, K., Landgren, B.M., Skjoldebrand-Sparre, L., Stavreus-Evers, A., Sundstrom-Poromaa, I., et al. 2014. The histidine-rich glycoprotein A1042G polymorphism and recurrent miscarriage: a pilot study. Reprod Biol Endocrinol 12:70.

125. Karehed, K., Wikstrom, A.K., Olsson, A.K., Larsson, A., Olovsson, M., and Akerud, H. 2010. Fibrinogen and histidine-rich glycoprotein in early-onset preeclampsia. Acta Obstet Gynecol Scand 89:131-139.

126. Bolin, M., Akerud, P., Hansson, A., and Akerud, H. 2011. Histidine-rich glycoprotein as an early biomarker of preeclampsia. Am J Hypertens 24:496-501.

127. Welsh, M., Mares, J., Karlsson, T., Lavergne, C., Breant, B., and Claesson-Welsh, L. 1994. Shb is a ubiquitously expressed Src homology 2 protein. Oncogene 9:19-27.

128. Anneren, C., Lindholm, C.K., Kriz, V., and Welsh, M. 2003. The FRK/RAK-SHB signaling cascade: a versatile signal-transduction pathway that regulates cell survival, differentiation and proliferation. Curr Mol Med 3:313-324.

129. Koch, C.A., Anderson, D., Moran, M.F., Ellis, C., and Pawson, T. 1991. SH2 and SH3 domains: elements that control interactions of cytoplasmic signaling proteins. Science 252:668-674.

130. Claesson-Welsh, L., Welsh, M., Ito, N., Anand-Apte, B., Soker, S., Zetter, B., O'Reilly, M., and Folkman, J. 1998. Angiostatin induces endothelial cell apoptosis and activation of focal adhesion kinase independently of the integrin-binding motif RGD. Proc Natl Acad Sci U S A 95:5579-5583.

131. Dixelius, J., Larsson, H., Sasaki, T., Holmqvist, K., Lu, L., Engstrom, A., Timpl, R., Welsh, M., and Claesson-Welsh, L. 2000. Endostatin-induced tyrosine kinase signaling through the Shb adaptor protein regulates endothelial cell apoptosis. Blood 95:3403-3411.

67

132. Cross, M.J., Lu, L., Magnusson, P., Nyqvist, D., Holmqvist, K., Welsh, M., and Claesson-Welsh, L. 2002. The Shb adaptor protein binds to tyrosine 766 in the FGFR-1 and regulates the Ras/MEK/MAPK pathway via FRS2 phosphorylation in endothelial cells. Mol Biol Cell 13:2881-2893.

133. Holmqvist, K., Cross, M.J., Rolny, C., Hagerkvist, R., Rahimi, N., Matsumoto, T., Claesson-Welsh, L., and Welsh, M. 2004. The adaptor protein shb binds to tyrosine 1175 in vascular endothelial growth factor (VEGF) receptor-2 and regulates VEGF-dependent cellular migration. J Biol Chem 279:22267-22275.

134. Lu, L., Holmqvist, K., Cross, M., and Welsh, M. 2002. Role of the Src homology 2 domain-containing protein Shb in murine brain endothelial cell proliferation and differentiation. Cell Growth Differ 13:141-148.

135. Funa, N.S., Kriz, V., Zang, G., Calounova, G., Akerblom, B., Mares, J., Larsson, E., Sun, Y., Betsholtz, C., and Welsh, M. 2009. Dysfunctional microvasculature as a consequence of shb gene inactivation causes impaired tumor growth. Cancer Res 69:2141-2148.

136. Lindholm, C.K., Gylfe, E., Zhang, W., Samelson, L.E., and Welsh, M. 1999. Requirement of the Src homology 2 domain protein Shb for T cell receptor-dependent activation of the interleukin-2 gene nuclear factor for activation of T cells element in Jurkat T cells. J Biol Chem 274:28050-28057.

137. Lindholm, C.K., Henriksson, M.L., Hallberg, B., and Welsh, M. 2002. Shb links SLP-76 and Vav with the CD3 complex in Jurkat T cells. Eur J Biochem 269:3279-3288.

138. Kriz, V., Agren, N., Lindholm, C.K., Lenell, S., Saldeen, J., Mares, J., and Welsh, M. 2006. The SHB adapter protein is required for normal maturation of mesoderm during in vitro differentiation of embryonic stem cells. J Biol Chem 281:34484-34491.

139. Gustafsson, K., Calounova, G., Hjelm, F., Kriz, V., Heyman, B., Gronvik, K.O., Mostoslavsky, G., and Welsh, M. 2011. Shb deficient mice display an augmented TH2 response in peripheral CD4+ T cells. BMC Immunol 12:3.

140. Karlsson, T., and Welsh, M. 1996. Apoptosis of NIH3T3 cells overexpressing the Src homology 2 domain protein Shb. Oncogene 13:955-961.

141. Welsh, M., Christmansson, L., Karlsson, T., Sandler, S., and Welsh, N. 1999. Transgenic mice expressing Shb adaptor protein under the control of rat insulin promoter exhibit altered viability of pancreatic islet cells. Mol Med 5:169-180.

142. Davoodpour, P., Landstrom, M., and Welsh, M. 2007. Reduced tumor growth in vivo and increased c-Abl activity in PC3 prostate cancer cells overexpressing the Shb adapter protein. BMC Cancer 7:161.

68

143. Kriz, V., Mares, J., Wentzel, P., Funa, N.S., Calounova, G., Zhang, X.Q., Forsberg-Nilsson, K., Forsberg, M., and Welsh, M. 2007. Shb null allele is inherited with a transmission ratio distortion and causes reduced viability in utero. Dev Dyn 236:2485-2492.

144. Calounova, G., Livera, G., Zhang, X.Q., Liu, K., Gosden, R.G., and Welsh, M. 2010. The Src homology 2 domain-containing adapter protein B (SHB) regulates mouse oocyte maturation. PLoS One 5:e11155.

145. Kriz, V., Anneren, C., Lai, C., Karlsson, J., Mares, J., and Welsh, M. 2003. The SHB adapter protein is required for efficient multilineage differentiation of mouse embryonic stem cells. Exp Cell Res 286:40-56.

146. Akerblom, B., Barg, S., Calounova, G., Mokhtari, D., Jansson, L., and Welsh, M. 2009. Impaired glucose homeostasis in Shb-/- mice. J Endocrinol 203:271-279.

147. Mokhtari, D., Kerblom, B., Mehmeti, I., Wang, X., Funa, N.S., Olerud, J., Lenzen, S., Welsh, N., and Welsh, M. 2009. Increased Hsp70 expression attenuates cytokine-induced cell death in islets of Langerhans from Shb knockout mice. Biochem Biophys Res Commun 387:553-557.

148. Okazaki, S., and Sue, S. 1995. Methodological issues in assessment research with ethnic minorities. Psychological Assessment 7:367 - 375.

149. Burgard, S.A., and Chen, P.V. 2014. Challenges of health measurement in studies of health disparities. Soc Sci Med 106:143-150.

150. Villanueva, E.V. 2001. The validity of self-reported weight in US adults: a population based cross-sectional study. BMC Public Health 1:11.

151. Chaves, R.N., de Matos, M.H., Buratini, J., Jr., and de Figueiredo, J.R. 2012. The fibroblast growth factor family: involvement in the regulation of folliculogenesis. Reprod Fertil Dev 24:905-915.

152. Porchet, H.C., le Cotonnec, J.Y., and Loumaye, E. 1994. Clinical pharmacology of recombinant human follicle-stimulating hormone. III. Pharmacokinetic-pharmacodynamic modeling after repeated subcutaneous administration. Fertil Steril 61:687-695.

153. van Santbrink, E.J., Hop, W.C., van Dessel, T.J., de Jong, F.H., and Fauser, B.C. 1995. Decremental follicle-stimulating hormone and dominant follicle development during the normal menstrual cycle. Fertil Steril 64:37-43.

154. Ben-Rafael, Z., Levy, T., and Schoemaker, J. 1995. Pharmacokinetics of follicle-stimulating hormone: clinical significance. Fertil Steril 63:689-700.

155. Oudendijk, J.F., Yarde, F., Eijkemans, M.J., Broekmans, F.J., and Broer, S.L. 2012. The poor responder in IVF: is the prognosis always poor?: a systematic review. Hum Reprod Update 18:1-11.

69

156. Hunault, C.C., Eijkemans, M.J., Pieters, M.H., te Velde, E.R., Habbema, J.D., Fauser, B.C., and Macklon, N.S. 2002. A prediction model for selecting patients undergoing in vitro fertilization for elective single embryo transfer. Fertil Steril 77:725-732.

157. Strandell, A., Bergh, C., and Lundin, K. 2000. Selection of patients suitable for one-embryo transfer may reduce the rate of multiple births by half without impairment of overall birth rates. Hum Reprod 15:2520-2525.

158. Hart, R., Khalaf, Y., Yeong, C.T., Seed, P., Taylor, A., and Braude, P. 2001. A prospective controlled study of the effect of intramural uterine fibroids on the outcome of assisted conception. Hum Reprod 16:2411-2417.

159. Fragouli, E., Wells, D., Iager, A.E., Kayisli, U.A., and Patrizio, P. 2012. Alteration of gene expression in human cumulus cells as a potential indicator of oocyte aneuploidy. Hum Reprod 27:2559-2568.

160. van Montfoort, A.P., Geraedts, J.P., Dumoulin, J.C., Stassen, A.P., Evers, J.L., and Ayoubi, T.A. 2008. Differential gene expression in cumulus cells as a prognostic indicator of embryo viability: a microarray analysis. Mol Hum Reprod 14:157-168.

161. Devjak, R., Fon Tacer, K., Juvan, P., Virant Klun, I., Rozman, D., and Vrtacnik Bokal, E. 2012. Cumulus cells gene expression profiling in terms of oocyte maturity in controlled ovarian hyperstimulation using GnRH agonist or GnRH antagonist. PLoS One 7:e47106.

162. Ambekar, A.S., Nirujogi, R.S., Srikanth, S.M., Chavan, S., Kelkar, D.S., Hinduja, I., Zaveri, K., Prasad, T.S., Harsha, H.C., Pandey, A., et al. 2013. Proteomic analysis of human follicular fluid: a new perspective towards understanding folliculogenesis. J Proteomics 87:68-77.

163. Klagsbrun, M., and D'Amore, P.A. 1991. Regulators of angiogenesis. Annu Rev Physiol 53:217-239.

164. Farach, M.C., Tang, J.P., Decker, G.L., and Carson, D.D. 1987. Heparin/heparan sulfate is involved in attachment and spreading of mouse embryos in vitro. Dev Biol 123:401-410.

165. Rohde, L.H., and Carson, D.D. 1993. Heparin-like glycosaminoglycans participate in binding of a human trophoblastic cell line (JAR) to a human uterine epithelial cell line (RL95). J Cell Physiol 155:185-196.

166. Revelli, A., Delle Piane, L., Casano, S., Molinari, E., Massobrio, M., and Rinaudo, P. 2009. Follicular fluid content and oocyte quality: from single biochemical markers to metabolomics. Reprod Biol Endocrinol 7:40.

70

167. Malamitsi-Puchner, A., Sarandakou, A., Baka, S.G., Tziotis, J., Rizos, D., Hassiakos, D., and Creatsas, G. 2001. Concentrations of angiogenic factors in follicular fluid and oocyte-cumulus complex culture medium from women undergoing in vitro fertilization: association with oocyte maturity and fertilization. Fertil Steril 76:98-101.

168. Barroso, G., Barrionuevo, M., Rao, P., Graham, L., Danforth, D., Huey, S., Abuhamad, A., and Oehninger, S. 1999. Vascular endothelial growth factor, nitric oxide, and leptin follicular fluid levels correlate negatively with embryo quality in IVF patients. Fertil Steril 72:1024-1026.

169. Bayasula, Iwase, A., Kobayashi, H., Goto, M., Nakahara, T., Nakamura, T., Kondo, M., Nagatomo, Y., Kotani, T., and Kikkawa, F. 2013. A proteomic analysis of human follicular fluid: comparison between fertilized oocytes and non-fertilized oocytes in the same patient. J Assist Reprod Genet 30:1231-1238.

170. Freeman, C., and Parish, C.R. 1997. A rapid quantitative assay for the detection of mammalian heparanase activity. Biochem J 325 ( Pt 1):229-237.

171. da Silveira, J.C., Veeramachaneni, D.N., Winger, Q.A., Carnevale, E.M., and Bouma, G.J. 2012. Cell-secreted vesicles in equine ovarian follicular fluid contain miRNAs and proteins: a possible new form of cell communication within the ovarian follicle. Biol Reprod 86:71.

172. Ottosen, L.D., Kesmodel, U., Hindkjaer, J., and Ingerslev, H.J. 2007. Pregnancy prediction models and eSET criteria for IVF patients--do we need more information? J Assist Reprod Genet 24:29-36.

173. Krussel, J.S., Bielfeld, P., Polan, M.L., and Simon, C. 2003. Regulation of embryonic implantation. Eur J Obstet Gynecol Reprod Biol 110 Suppl 1:S2-9.

174. Spiro, R.G. 2002. Protein glycosylation: nature, distribution, enzymatic formation, and disease implications of glycopeptide bonds. Glycobiology 12:43R-56R.

175. Dixelius, J., Olsson, A.K., Thulin, A., Lee, C., Johansson, I., and Claesson-Welsh, L. 2006. Minimal active domain and mechanism of action of the angiogenesis inhibitor histidine-rich glycoprotein. Cancer Res 66:2089-2097.

176. Ignjatovic, V., Lai, C., Summerhayes, R., Mathesius, U., Tawfilis, S., Perugini, M.A., and Monagle, P. 2011. Age-related differences in plasma proteins: how plasma proteins change from neonates to adults. PLoS One 6:e17213.

177. Drasin, T., and Sahud, M. 1996. Blood-type and age affect human plasma levels of histidine-rich glycoprotein in a large population. Thromb Res 84:179-188.

178. Jones, A.L., Hulett, M.D., and Parish, C.R. 2004. Histidine-rich glycoprotein binds to cell-surface heparan sulfate via its N-terminal domain following Zn2+ chelation. J Biol Chem 279:30114-30122.

71

179. Martinez-Hernandez, M.G., Baiza-Gutman, L.A., Castillo-Trapala, A., and Armant, D.R. 2011. Regulation of proteinases during mouse peri-implantation development: urokinase-type plasminogen activator expression and cross talk with matrix metalloproteinase 9. Reproduction 141:227-239.

180. Carroll, P.M., Richards, W.G., Darrow, A.L., Wells, J.M., and Strickland, S. 1993. Preimplantation mouse embryos express a cell surface receptor for tissue-plasminogen activator. Development 119:191-198.

181. Axelrod, H.R. 1985. Altered trophoblast functions in implantation-defective mouse embryos. Dev Biol 108:185-190.

182. Silverstein, R.L., Leung, L.L., Harpel, P.C., and Nachman, R.L. 1984. Complex formation of platelet thrombospondin with plasminogen. Modulation of activation by tissue activator. J Clin Invest 74:1625-1633.

183. Stein, J.L., Hua, X., Lee, S., Ho, A.J., Leow, A.D., Toga, A.W., Saykin, A.J., Shen, L., Foroud, T., Pankratz, N., et al. 2010. Voxelwise genome-wide association study (vGWAS). Neuroimage 53:1160-1174.

184. Palmieri, F. 2013. The mitochondrial transporter family SLC25: identification, properties and physiopathology. Mol Aspects Med 34:465-484.

185. Pritchard, D.J., and Korf, B.R. 2013. Medical genetics at a glance. Oxford: Wiley-Blackwell.

186. Welsh, M., Anneren, C., Lindholm, C., Kriz, V., and Oberg-Welsh, C. 2000. Role of tyrosine kinase signaling for beta-cell replication and survival. Ups J Med Sci 105:7-15.

187. Anneren, C., and Welsh, M. 2002. GTK tyrosine kinase-induced alteration of IRS-protein signalling in insulin producing cells. Mol Med 8:705-713.

188. Hansen, K.R., Hodnett, G.M., Knowlton, N., and Craig, L.B. 2011. Correlation of ovarian reserve tests with histologically determined primordial follicle number. Fertil Steril 95:170-175.

189. Blazquez, A., Guillen, J.J., Colome, C., Coll, O., Vassena, R., and Vernaeve, V. 2014. Empty follicle syndrome prevalence and management in oocyte donors. Hum Reprod.

190. Gonca, S., Gun, I., Ovayolu, A., Silfeler, D., Sofuoglu, K., Ozdamar, O., Yilmaz, A., and Tunali, G. 2014. Effect of lower than expected number of oocyte on the IVF results after oocyte-pickup. Int J Clin Exp Med 7:1853-1859.

191. Chorev, M., and Carmel, L. 2012. The function of introns. Front Genet 3:55.

192. Redmer, D.A., and Reynolds, L.P. 1996. Angiogenesis in the ovary. Rev Reprod 1:182-192.

193. McFee, R.M., and Cupp, A.S. 2013. Vascular contributions to early ovarian development: potential roles of VEGFA isoforms. Reprod Fertil Dev 25:333-342.

72

194. Christoffersson, G., Zang, G., Zhuang, Z.W., Vagesjo, E., Simons, M., Phillipson, M., and Welsh, M. 2012. Vascular adaptation to a dysfunctional endothelium as a consequence of Shb deficiency. Angiogenesis 15:469-480.

195. McFee, R.M., Artac, R.A., Clopton, D.T., Smith, R.A., Rozell, T.G., and Cupp, A.S. 2009. Inhibition of vascular endothelial growth factor receptor signal transduction blocks follicle progression but does not necessarily disrupt vascular development in perinatal rat ovaries. Biol Reprod 81:966-977.

196. Zang, G., Christoffersson, G., Tian, G., Harun-Or-Rashid, M., Vagesjo, E., Phillipson, M., Barg, S., Tengholm, A., and Welsh, M. 2013. Aberrant association between vascular endothelial growth factor receptor-2 and VE-cadherin in response to vascular endothelial growth factor-a in Shb-deficient lung endothelial cells. Cell Signal 25:85-92.

197. Gustafsson, K., Heffner, G., Wenzel, P.L., Curran, M., Grawe, J., McKinney-Freeman, S.L., Daley, G.Q., and Welsh, M. 2013. The Src homology 2 protein Shb promotes cell cycle progression in murine hematopoietic stem cells by regulation of focal adhesion kinase activity. Exp Cell Res 319:1852-1864.

198. McGinnis, L.K., Carroll, D.J., and Kinsey, W.H. 2011. Protein tyrosine kinase signaling during oocyte maturation and fertilization. Mol Reprod Dev 78:831-845.

199. Meng, L., Luo, J., Li, C., and Kinsey, W.H. 2006. Role of Src homology 2 domain-mediated PTK signaling in mouse zygotic development. Reproduction 132:413-421.

200. Sette, C., Paronetto, M.P., Barchi, M., Bevilacqua, A., Geremia, R., and Rossi, P. 2002. Tr-kit-induced resumption of the cell cycle in mouse eggs requires activation of a Src-like kinase. EMBO J 21:5386-5395.

201. Talmor-Cohen, A., Tomashov-Matar, R., Tsai, W.B., Kinsey, W.H., and Shalgi, R. 2004. Fyn kinase-tubulin interaction during meiosis of rat eggs. Reproduction 128:387-393.

202. Besterman, B., and Schultz, R.M. 1990. Regulation of mouse preimplantation development: inhibitory effect of genistein, an inhibitor of tyrosine protein phosphorylation, on cleavage of one-cell embryos. J Exp Zool 256:44-53.

203. Jacquet, P., de Saint-Georges, L., Barrio, S., and Baugnet-Mahieu, L. 1995. Morphological effects of caffeine, okadaic acid and genistein in one-cell mouse embryos blocked in G2 by X-irradiation. Int J Radiat Biol 67:347-358.

204. Gong, J.G., McBride, D., Bramley, T.A., and Webb, R. 1993. Effects of recombinant bovine somatotrophin, insulin-like growth factor-I and insulin on the proliferation of bovine granulosa cells in vitro. J Endocrinol 139:67-75.

73

205. Wang, H.S., and Chard, T. 1999. IGFs and IGF-binding proteins in the regulation of human ovarian and endometrial function. J Endocrinol 161:1-13.

206. Nelson, S.M., Yates, R.W., Lyall, H., Jamieson, M., Traynor, I., Gaudoin, M., Mitchell, P., Ambrose, P., and Fleming, R. 2009. Anti-Mullerian hormone-based approach to controlled ovarian stimulation for assisted conception. Hum Reprod 24:867-875.

207. Yates, A.P., Roberts, S.A., and Nardo, L.G. 2012. Anti-Mullerian hormone-tailored stimulation protocols improve outcomes whilst reducing adverse effects and costs of IVF. Hum Reprod 27:629.

208. Popovic-Todorovic, B., Loft, A., Lindhard, A., Bangsboll, S., Andersson, A.M., and Andersen, A.N. 2003. A prospective study of predictive factors of ovarian response in 'standard' IVF/ICSI patients treated with recombinant FSH. A suggestion for a recombinant FSH dosage normogram. Hum Reprod 18:781-787.

209. Botros, L., Sakkas, D., and Seli, E. 2008. Metabolomics and its application for non-invasive embryo assessment in IVF. Mol Hum Reprod 14:679-690.

Acta Universitatis UpsaliensisDigital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Medicine 1048

Editor: The Dean of the Faculty of Medicine

A doctoral dissertation from the Faculty of Medicine, UppsalaUniversity, is usually a summary of a number of papers. A fewcopies of the complete dissertation are kept at major Swedishresearch libraries, while the summary alone is distributedinternationally through the series Digital ComprehensiveSummaries of Uppsala Dissertations from the Faculty ofMedicine. (Prior to January, 2005, the series was publishedunder the title “Comprehensive Summaries of UppsalaDissertations from the Faculty of Medicine”.)

Distribution: publications.uu.seurn:nbn:se:uu:diva-234067

ACTAUNIVERSITATIS

UPSALIENSISUPPSALA

2014