Background Animal models can be used to mimic human conditions of psychopathology, and also as pre-clinical models to evaluate candidate drugs. With hormonal treatment it is possible to produce behavior in the rat which corresponds to the mental symptoms of pre-menstrual syndrome (PMS), and pre-menstrual dysphoric disorder (PMDD). PMS affects 25-30 % of all women in fertile age and 3-8% are diagnosed with the more severe condition PMDD. The cardinal mental symptoms are; irritability, mood-swings, depression, anxiety, fatigue, insomnia, difficulties with concentration and memory and learning difficulties. The symptoms of PMS/PMDD occur in the luteal phase in conjunction with increasing concentrations of progesterone (P4) and P4-metabolites. In anovulatory cycles the symptoms are absent. The hormones which produce the monthly reoccurring negative symptoms on mood are foremost the neuroactive metabolites; allopregnanolone (ALLO) and tetrahydro-deoxycorticosterone (THDOC). ALLO is produced by the corpus luteum, but can also be synthesized in the brain, both ALLO and THDOC can also be released from the adrenal cortex during stress. These steroids are active on the inhibitory GABA neurotransmitter system through the GABAA receptor, and the effects are similar to that of alcohol and benzodiazepines. These steroids have strong sedative and hypnotic effects. A paradox is that some individuals seem to react with negative mood on sex steroids while all fertile women have the cyclical steroid changes during the menstrual cycle. Some individuals are more sensitive to neuroactive steroids with influences of personality, heritability and stress factors. Aims The thesis aims were to develop pre-clinical animal models of PMS/PMDD and to investigate induction of ALLO tolerance, individual sensitivity to neurosteroids and the interactions between chronic social stress and neurosteroids. Methods In these studies male and female Wistar rats were used to test steroid hormone effects on learning and memory and behaviors analogous to negative mood symptoms. This was accomplished through hormonal treatment and a subsequent withdrawal period from P4 (P4) + estradiol (E2) (PEWD), or ALLO. To assess tolerance, memory and learning in the Morris water maze (MWM) was studied. Anxiety-like behaviors were tested with the elevated plus maze (EPM), open field test (OFT), and the intruder test (IT). The EPM or OFT was used to classify the rats as high or low responders on risk-taking and explorative behavior (HR/LR). For social ranking order assessment the tube test (TT) and food competition test (FCT) were used. Chronic social stress was accomplished through co-habituation with two older rats (chronic subordination stress). In female rats the estrous cycle followed using staining of vaginal smears. Concentration of corticosterone (CORT) was measured by radio-immuno-assay (RIA). Results In the MWM ALLO pre-treatment produced tolerance to the acute negative ALLO effects. Both male and female rats showed behavioral correlations between the EPM and OFT tests, and correlations were also seen in CORT levels. Individuals with the stable trait of high risk-taking and explorative behavior (HR) were more sensitive to PEWD induction of anxiety-like behavior. These animals also showed decreased CORT levels during withdrawal. Chronic subordination stress enhanced the response to PEWD on measures of locomotor activity and social anxiety-like behavior. Conclusions It is possible to induce tolerance to the negative ALLO effects on learning and memory. The animal models of anxiety-like behavior show an individual PEWD response profile where HR rats are more sensitive. Exposure to chronic social stress enhanced the PEWD response. Hence there are both inherent and environmental factors behind the behavioral response to steroid hormones in rats. / Stress- och könshormoners verkningar på centrala nervsystemet

Many women experience miscarriage every year. Every fourth woman who has given birth reports that she has previous experience of miscarriage. In a study of all women in the Swedish Medical Birth Register 1983-2003, we found that the number of cases of self reported miscarriage had increased in Sweden during this 21 year period. This increase can be explained by the introduction of sensitive pregnancy tests around 1990, as well as an increase in the mean age of the mothers, by approximately 3 years, during the observation period. The risk of miscarriage is 13% with the first child. With subsequent pregnancies, the risk of miscarriage is 8%, 6% and 4% with the second, third and fourth child, respectively. Thirteen of these women who had suffered a recent miscarriage were interviewed four months later, and their feelings of guilt and emptiness were explored. Their experience was that they wanted their questions to be answered, and that they wanted others to treat them as the mothers to be that they felt themselves to be. They also experienced the need for time to grieve their loss. Measurement of grief by means of the Perinatal Grief Scale (PGS) is used in research but has also been proposed for clinical use. We have translated this psychological instrument to Swedish, back-translated and tested it in a small pilot study. In a randomized controlled study, women with early miscarriage were allocated, either to a structured visit (study group) or a regular visit (control group) to a midwife. The structured visit was conducted according to the Swanson caring theory. We could conclude that the structured visit had no significant effect on grief compared to the regular visit, as measured using the PGS. However, women with the sub-diagnosis missed abortion have significantly more grief four months after early miscarriage, regardless of visit type. We also performed a content analysis of the tape-recorded structured follow-up visit. The code-key used was Bonanno and Kaltman’s general grief categorization. Women’s expression of grief after miscarriage was found to be very similar to the grief experienced following the death of a relative. Furthermore, the grief was found to be independent of number of children, women’s age, or earlier experience of miscarriage. Conclusions: Every fourth woman who gives birth reports that she has also experienced early miscarriage. The experience of these women is that they have suffered a substantial loss and their reaction is grief similar to that experienced following the death of a relative. / On the day of the public defence of the doctoral thesis the status of article III was In Press and article IV was In Press.

In the past decades there has been an improvement in the medical treatment of children born preterm or with reduced foetal growth. This has resulted in a much higher survival rate of these children, but also in a higher number of surviving children with chronic conditions. These changes have, in turn, increased interest in investigating the connection between birth-characteristics and outcomes in later life. The overall aim of the present thesis was to study the relations between birth-characteristics, subsequent hospitalisations, and childbearing by means of data available in Swedish population-based registries. The study population in this thesis consisted of women (and men in Paper III) born in 1973-75 according to the Medical Birth Register and the Total Population Register. Information available in other registries, such as the Hospital Discharge Register, was obtained by individual record linkage. In Paper I, 148,281 women, alive and living in Sweden at 13 years of age, were included. Of the women, 4.1% were born preterm and 5.4% were born small for gestational age, and approximately 30% of all women had given birth between 13 and 27 years of age. We found that reduced foetal growth and possibly preterm birth were related to the likelihood of giving birth during the study period. The intergenerational effects of preterm birth and reduced foetal growth were investigated in Paper II and the study population consisted of 38,720 mother-offspring pairs. An intergenerational effect of reduced foetal growth was found, and reduced foetal growth in the mother also increased the risk for preterm birth in the child. Paper III was concerned with 304,275 men and women living in Sweden at 13 years of age. Of these men and women, 30% were hospitalised during adolescence and early adulthood (i.e. between 12 and 23 years of age). We found that men and women born small for gestational age or preterm were more likely to be hospitalised, and that those born small for gestational age seemed to be more at risk compared to those born preterm. Finally, in Paper IV, the relation between hospitalisations during adolescence and the likelihood of giving birth was studied in 142,998 women living in Sweden at 20 years of age. We found that a majority of the causes of hospitalisation during adolescence were positively connected to the likelihood of giving birth between 20 and 27 years of age. The relations presented in Papers I-IV were evident although socio-economic characteristics were adjusted for.

Progesterone metabolites as allopregnanolone, isoallopregnanolone and tetrahydrodeoxy-corticosterone (THDOC) are increased in the luteal phase of the menstrual cycle, throughout pregnancy and during stress. Allopregnanolone and THDOC are neurosteroids with 3α-hydroxy, 5α-configurations and positive modulating effect on the GABAA receptor. They have similar properties and effect, and share the same binding sites on the GABAA receptor. Isoallopregnanolone has a 3β-hydroxy, 5α-configuration and a diverse effect as a proposed antagonist to both allopregnanolone and THDOC. Neurosteroids are thought to exert their effect predominantly at extrasynaptic GABAA receptors, containing for example α4- or α5-subunits. Such receptors are involved in the tonic response. Different subunits have diverse distribution pattern in the brain and are involved in different functions. The α5-subunit, mainly expressed in the hippocampus, is involved in learning, while α4 is more widespread and involved in e.g. anxiety and anaesthesia. The aim of the present thesis was to contribute to the knowledge about selected progesterone metabolites and their effects on learning and tolerance development, as well as their metabolism. Also basic characteristics between different α-subunits of the GABAA receptor were evaluated. The thesis shows that the effect of bicuculline and pentobarbital is not dependent on the α-subunit isoform of the GABAA receptor expressed in oocytes. Acute tolerance developed after allopregnanolone-induced anaesthesia with a decrease at both mRNA and protein levels of the GABAA receptor α4-subunit in the thalamus VPM nucleus. A negative correlation between the α4 mRNA and the increased dose of allopregnanolone needed to maintain the anaesthesia level was also shown. In addition, allopregnanolone induces a learning impairment in the Morris water maze test, when high concentrations of allopregnanolone are present in the brain. This impairment is not possible to reverse by isoallopregnanolone. In α5β3γ2L-transfected HEK-293 cells THDOC induces a baseline shift of its own and also potentiate the GABA-current. Neither of those THDOC effects can be inhibited by isoallopregnanolone. Instead isoallopregnanolone shows an agonistic effect on the THDOC-potentiation of the GABA-response. The main allopregnanolone metabolites identified, 5α-DHP and isoallopregnanolone, as well as allopregnanolone itself are mainly localized to the brain after an i.v. injection. After an isoallopregnanolone injection there is a more even distribution of the given steroid and the metabolites between plasma and brain. There is an epimerisation between isoallopregnanolone and allopregnanolone and vice versa. In conclusion, the present thesis shows that the α4-subunit in the thalamus VPM nucleus is likely to be involved in the acute tolerance development against allopregnanolone and that allopregnanolone-induced learning impairment is likely to be hippocampus dependent. The lack of antagonistic effect of isoallopregnanolone on the THDOC-induced α5β3γ2L-GABAA response, together with epimerisation of isoallopregnanolone to allopregnanolone, could explain why isoallopregnanolone does not work as an antagonist to the allopregnanolone-induced learning impairment in a hippocampus dependent learning task.

Background: Chlamydia trachomatis in women is a risk factor for tubal factor infertility and extra uterine pregnancies, but the impact of a C. trachomatis infection on male fertility is unclear. It is also hypothesized that persistent infection with C. trachomatis, or other microorganisms, might initiate/promote ovarian tumor development. The aims of the thesis were to study whether C. trachomatis serum antibodies in women and men had an impact on infertility diagnoses, semen characteristics, pregnancy rates and pregnancy outcomes; furthermore, to explore associations of C. trachomatis, and Mycoplasma genitalium, plasma antibodies with epithelial ovarian cancer and borderline ovarian tumors, as well as the presence of C. trachomatis bacteria, and other microorganisms, in ovarian tissues. Materials and methods: Papers I and II: 244/226 infertile couples were tested for serum C. trachomatis IgG, IgA, IgM and chlamydial Heat Shock Protein 60 (cHSP60) IgG antibodies. C. trachomatis IgG positive couples were also tested for C. trachomatis DNA in a urine sample. The follow-up period was 14-54 months. 244 spontaneously pregnant women were also tested for serum C. trachomatis IgG antibodies. Papers III and IV: Plasma samples from 291 women with epithelial ovarian cancer, borderline ovarian tumors and benign conditions, and plasma samples from 271 healthy controls, were analyzed for C. trachomatis IgG, IgA and cHSP60-1 IgG and M. genitalium IgG antibodies. Ovarian tissues from 186 women with benign ovaries, borderline ovarian tumors and epithelial ovarian cancer, as well as tissues from the contra lateral ovary in 126 women, were analyzed for the presence of C. trachomatis, M. genitalium, Neisseria gonorrhoeae, HPV and the polyoma viruses BKV and JCV with nucleic acid amplification tests. Results: Papers I and II: The prevalence of C. trachomatis IgG antibodies was higher among infertile than fertile women, and there were 9 couples with ongoing C. trachomatis infections. In men, C. trachomatis IgG and IgA antibodies were associated with a reduced likelihood to achieve pregnancy for the couple, as well as lower sperm concentration, reduced sperm motility and vitality, increased teratozoospermia index and the occurrence of leukocytes. C. trachomatis IgG and cHSP60 IgG antibodies in infertile women were associated with tubal factor infertility, but not with reduced pregnancy rates or outcomes. Paper III: cHSP60-1 IgG antibodies were associated with ovarian cancer belonging to the postulated type II pathogenetic pathway when plasma samples obtained more than one year prior to diagnosis were analyzed. M. genitalium IgG antibodies were associated with borderline ovarian tumors; however a statistical type 1 error cannot be excluded. Paper IV: None of the microorganisms studied were found in the ovarian tissue samples. Conclusions: C. trachomatis IgG and IgA antibodies in the man substantially decreases the chances of the infertile couple to achieve pregnancy, and are associated with subtle negative changes in semen characteristics. C. trachomatis IgG and cHSP60 IgG antibodies in the woman are risk factors for tubal factor infertility. Prospective plasma cHSP60-1 IgG antibodies are associated with type II ovarian carcinomas, but C. trachomatis bacteria, or the other microorganisms studied, could not be detected in benign, borderline or malignant ovarian tissues.

Aim: To investigate gynecologists’ and midwives’ views and experiences regarding work in abortion care in Sweden. Methods: Questionnaire to gynecologists (n=269) and midwives (n=258 comprising 48 questions, response 85%. The quantitative studies (articles I-III) were supplemented by a qualitative study (article IV), consisting of focus-group interviews with gynecologists and midwives/nurses. Results: From the questionnaire studies it was apparent that all the gynecologists had worked in abortion care, whilst not all midwives had done so. The male gynecologists were older than both their female colleagues and the midwives; they had most years of experience but were now working least with abortion patients. Both groups considered it absolutely right, that Sweden have legal abortion and that the law was being followed. Most thought that women should be allowed to have an abortion even after they had felt fetal movements. The midwives were generally somewhat more restrictive than the gynecologists. Half of all thought that the work with abortion patients brought something positive with it. Those having worked longest and most extensively, especially during the previous year were most liberal. Both groups felt that there was a difference between working with surgical and late abortions compared with medical abortions. One in four had had misgivings when involved in surgical and medical abortions, and one in two with abortions after the 18th week. All were positive about the transition to medical abortions, and roughly two thirds of the midwives thought that the primary care sector should be able to take care of these, whereas less than half of the gynecologists thought this. The majority considered it important to receive further and continuing professional development and ongoing guidance. From the focus-group interviews it was clear that the experiences of the gynecologists were largely connected with the technical development of abortion methods and those of the midwives/nurses with improved pain relief. The work was sometimes described in paradoxical terms and was occasionally experienced as frustrating, especially in connection with repeat abortions. Neither of the two groups, however, had had any doubts about participating in abortion. The gynecologists described how women now expected to get an abortion, whereas previously they had asked for one. The midwife/nurse group maintained that the meetings with the women had become considerably more frequent. The interaction between the two professional groups was marked by great trust in each other’s professional competence. Conclusions: Gynecologists and midwives working in abortion care support Swedish abortion legislation and have no doubts about participating in abortions, despite the fact that they have frequently experienced complex and difficult work situations. The character of the work is experienced as contradictory and frustrating, but also as challenging and rewarding. The awareness that the two professional groups have of the importance of continuing professional development and ongoing guidance should be acted on. Furthermore, their collective views and experiences should be made use of, so that abortion care can be developed, not only in order to promote women’s health, but also to improve the work environment for the abortion staff.

This thesis aims to study the effects of endogenous and exogenous hormones and mammographic breast density (BD) on cellular markers in non-cancerous female breast tissue. Women on the waiting list for breast reduction plastic surgery were recruited (n = 79), and randomized to 2 months of hormone therapy or no therapy before surgery. The women had a mammogram and a needle biopsy 2 months before surgery and tissue samples were obtained at the operation. In premenopausal women, estrogen receptor (ER)α levels were associated with age (p = 0.0002), were similar in the follicular and luteal phases of the menstrual cycle and were higher in parous than in nulliparous women (p = 0.009). Current smokers had lower PR levels than non-smokers (p = 0.019). Women on oral contraception had lower ERα (p = 0.048) and PR (p = 0.007) levels than women in the follicular phase. The ERα levels did not differ significantly between postmenopausal estrogen and estrogen-progestogen users, but PR levels were lower among estrogen-progestogen users (p = 0.03). We found lower expression of the genes for decorin and syndecans 1 and 4 in the luteal phase than in the follicular phase, among parous women. Protein levels of the androgen receptor, syndecan-4 and decorin was lower in premenopausal women who were using oral contraceptives (OC) than in those in the follicular phase (p = 0.002 - 0.02), whereas no significant differences between OC use and the luteal phase were found. In premenopausal women, BD was negatively associated with age and body mass index but was similar for the menstrual phases. Breast density was associated with genetic expression of the androgen receptor and remained significant after adjustment for age (rs = 0.56; p = 0.04). After adjustement for age, breast density was also marginally associated with expression of the caspase 3 gene (0.55; 0.053). However, protein levels of caspase 3 was negatively associated (-0.61; 0.03).

Many studies have suggested a relationship between sex steroids and negative mental and mood changes in women. Allopregnanolone, a potent endogenous ligand of the GABA-A receptor and a metabolite of progesterone, is one of the most accused neuroactive steroids. Variations in the levels of neuroactive steroids that influence the activity of the GABA-A receptor cause a vulnerability to mental and emotional pathology. In women, there are physiological conditions in which allopregnanolone production increases acutely (e.g. stress) or chronically (e.g. menstrual cycle, pregnancy), thus exposing the GABA-A receptor to high allopregnanolone concentrations. In such conditions, tolerance to allopregnanolone probably develops. We have evaluated the 3β-hydroxy pregnane steroid UC1011 as a functional antagonist to allopregnanolone-induced negative effects in rats. In vivo, we used the Morris Water Maze (MWM) test of learning and, in vitro, we studied chloride ion uptake into cortical and hippocampal membrane preparations. The steroid UC1011 reduces the allopregnanolone-induced learning impairment in the MWM and the increase in chloride ion uptake induced by allopregnanolone. To detect whether chronic tolerance develops to an allopregnanolone-induced condition, male rats were pretreated with allopregnanolone injections for three or seven days. These rats were then tested in the Morris Water Maze for five days and compared with relevant controls. Rats with seven days’ allopregnanolone pretreatment experienced improved performance compared with the acutely allopregnanolone-exposed group, reflecting chronic tolerance development. To study the GABA-A receptor changes in acute allopregnanolone tolerance, we used the silent second (SS) anaesthesia threshold method. At acute tolerance, 90 minutes of anaesthesia, the abundance of the GABA-A receptor α4 subunit and the expression of the α4 subunit mRNA in the thalamus ventral-posteriomedial (VPM) nucleus were reduced. There was also a significant negative correlation between the increase in the allopregnanolone dose needed to maintain anaesthesia and the α4 mRNA in the VPM nucleus. We also investigated whether allopregnanolone tolerance was still present one or two days after the end of the anaesthesia-induced acute tolerance. Tolerance persisted to one day, but not two days, after the treatment and the α4 subunit mRNA expression in the VPM nucleus was negatively related to the allopregnanolone doses needed after one day. In conclusion, the current thesis shows that the substance UC1011 can reduce the allopregnanolone-induced negative effects in the water maze test. Chronic allopregnanolone tolerance can develop to the effects of allopregnanolone. Allopregnanolone tolerance persists one day after the induction of acute allopregnanolone tolerance. The GABA-A receptor α4 subunit in the thalamus might be involved in the development and persistence of acute tolerance to allopregnanolone.

Many young women today are concerned about their sexual health; an increasing number of them consult gynaecologists, youth centres (YCs) and general practitioners with vulvar problems such as painful sensations associated with vaginal intercourse (VIC). It is known that some women continue to have VIC despite pain. Theoretically, repeated painful VIC might elicit vaginistic reactions, which may increase the pain and induce vicious circles. Since many clinicians and researchers nowadays notice that pain during VIC often starts at young age, it is important to investigate how pain during VIC starts and is maintained in younger populations. The overall aim of this thesis was to investigate young women’s experiences of ideal sexual situations and pain during VIC. Women aged 13-22 years participated in our studies, which used both quantitative (study I and IV) and qualitative (study II and III) methods. For paper I, a questionnaire was developed and used in a YC sample (n=300); informants for paper II were selected from that sample to participate in qualitative interviews (n=16). Another qualitative interview study for paper III with a complimentary research question was conducted in a different YC sample (n=14). For paper IV, a questionnaire was developed based on the results from study I, II and III to test the hypotheses derived from study II in a sample of female high school students (n=1566). The findings revealed that 65% of the women reported pain related to first VIC. Among those who reported VIC during the previous month, 49% had experienced pain and/or discomfort during VIC during that same period (paper I). In paper IV, 47% of the women reported experience of pain and/or discomfort during VIC, and among those, 47% continued to have VIC, 22% feigned enjoyment, and 33% omitted telling the partner about their pain. In paper II, the women’s reasons for continuing to have VIC despite pain were: striving to reach their ideal image of a woman, characterized as always willing to have VIC; being perceptive of their partner’s sexual needs; and being able to satisfy their partner. In paper IV the hypotheses derived from study II were confirmed and showed, for example that a significantly higher proportion of women who continue to have VIC despite pain than women who did not had difficulty refusing sex when the partner wants it, felt inferior to the partner during sex, regarded the partner’s satisfaction as more important than their own, felt dissatisfaction with their sex life, and feigned enjoyment despite pain. In a multivariate model, continuing to have VIC despite pain was associated with feelings of being inferior to the partner during sex (adjusted OR 1.82; CI 1.10-3.02), dissatisfaction with their own sex lives (adjusted OR 1.76; CI 1.14-2.72) and feigning enjoyment while having pain (adjusted OR 7.45; CI 4.37-12.69). The major reason for continuing to have VIC was that the partner’s enjoyment was prioritized higher than their own (paper IV). In paper III, we found that women without pain during VIC also felt pressure from social norms and demands and had experienced partners “driving their own race”. However, they managed to some extent to resist these unequal gender norms because of their urge to experience pleasure. In conclusion, pain during VIC is a common complaint among young Swedish women, and a high proportion of them continue having VIC despite pain. The women’s notion of prioritizing the partners´ enjoyment before their own illustrates that unequal gender regimes affect young women’s (hetero)sexuality negatively.

Ovarian steroid hormones are neuroactive steroids with widespread actions in the brain, and are thus able to influence mood, behavior and cognition. In this thesis the effects of progesterone withdrawal and the direct effects of the progesterone metabolite allopregnanolone are evaluated. Allopregnanolone, through binding to the GABAA receptor complex, enhances inhibitory neurotransmission, thus exerting anxiolytic, sedative and antiepileptic effects. The acoustic startle response (ASR) is a withdrawal reflex evoked by sudden or noxious auditory stimuli, and can be measured in humans as an eye blink. ASR is significantly increased in several anxiety disorders, and notably also during progesterone withdrawal. Sensorimotor gating can be assessed by measuring prepulse inhibition of the startle response (PPI). The CNS circuits regulating PPI are sensitive to hormone fluctuations. GABAergic drugs are involved in cognitive impairment and animal studies have indicated that allopregnanolone may inhibit learning. The main purpose of this research was to evaluate the behavioral effects of progesterone withdrawal on the startle response and sensorimotor gating in PMDD patients and healthy controls, in healthy third trimester pregnant women and healthy postpartum women. A second aim was to evaluate allopregnanolone effects on memory and cognition in healthy women and also on the startle response and PPI. We found that PMDD patients have an increased startle response across the menstrual cycle and a deficiency in sensorimotor gating during the late luteal phase. Ovarian steroids affect sensorimotor gating; pregnant women have lower levels of PPI than late postpartum women. Acutely administered allopregnanolone did not affect the ASR or PPI. Allopregnanolone impairs episodic memory in healthy women. In conclusion, our studies suggest that ovarian steroids, including allopregnanolone, do not influence the startle response. Ovarian steroids affect sensorimotor gating; pregnancy, a condition with high levels of ovarian steroids, suppresses PPI. Theoretically, the variability in PPI across reproductive events is due to effects mediated by the progesterone or estradiol receptors but is not mediated by allopregnanolone. PMDD patients display decreased PPI during the late luteal phase, suggesting underlying pathophysiology in common with other anxiety disorders. The most vulnerable memory system, the episodic memory, is impaired by the allopregnanolone in healthy women.