Low androgen signaling rescues genome integrity with innate immune response by reducing fertility in humans.

Development of the gonads under complex androgen regulation is critical for germ cells specification. In this work we addressed the relationship between androgens and genomic integrity determining human fertility. We used different study groups: individuals with Differences of Sex Development (DSD), including Complete Androgen Insensitivity Syndrome (CAIS) due to mutated androgen receptor (AR), and men with idiopathic nonobstructive azoospermia. Both showed genome integrity status influenced by androgen signaling via innate immune response activation in blood and gonads. Whole proteome analysis connected low AR to interleukin-specific gene expression, while compromised genome stability and tumorigenesis were also supported by interferons. AR expression was associated with predominant DNA damage phenotype, that eliminated AR-positive Sertoli cells as the degeneration of gonads increased. Low AR contributed to resistance from the inhibition of DNA repair in primary leukocytes. Downregulation of androgen promoted apoptosis and specific innate immune response with higher susceptibility in cells carrying genomic instability.

Comparing gene expression in deep infiltrating endometriosis with adenomyosis uteri: evidence for dysregulation of oncogene pathways.

BACKGROUND: The pathogenesis of deep infiltrating endometriosis (DIE) is poorly understood. It is considered a benign disease but has histologic features of malignancy, such as local invasion or gene mutations. Moreover, it is not clear whether its invasive potential is comparable to that of adenomyosis uteri (FA), or whether it has a different biological background. Therefore, the aim of this study was to molecularly characterize the gene expression signatures of both diseases in order to gain insight into the common or different underlying pathomechanisms and to provide clues to pathomechanisms of tumor development based on these diseases. METHODS: In this study, we analyzed formalin-fixed and paraffin-embedded tissue samples from two independent cohorts. One cohort involved 7 female patients with histologically confirmed FA, the other cohort 19 female patients with histologically confirmed DIE. The epithelium of both entities was microdissected in a laser-guided fashion and RNA was extracted. We analyzed the expression of 770 genes using the nCounter expression assay human PanCancer (Nanostring Technology). RESULTS: In total, 162 genes were identified to be significantly down-regulated (n = 46) or up-regulated (n = 116) in DIE (for log2-fold changes of < 0.66 or > 1.5 and an adjusted p-value of < 0.05) compared to FA. Gene ontology and KEGG pathway analysis of increased gene expression in DIE compared to FA revealed significant overlap with genes upregulated in the PI3K pathway and focal adhesion signaling pathway as well as other solid cancer pathways. In FA, on the other hand, genes of the RAS pathway showed significant expression compared to DIE. CONCLUSION: DIE and FA differ significantly at the RNA expression level: in DIE the most expressed genes were those belonging to the PI3K pathway, and in FA those belonging to the RAS pathway.

'A sorrow shared ': a qualitative content analysis of what couples with recurrent miscarriages expect from one another and their families and friends.

STUDY QUESTION: When couples have to face recurrent pregnancy loss (RPL), what are the partners' wishes and needs and what is their perception of helpful and unhelpful factors with regard to their own, their partners' and their families' and friends' ways of dealing with the problem? SUMMARY ANSWER: Women and men with repeated miscarriages want open communication about their losses, but expect a sensitive and empathetic attitude from others, not pity or trivialization. WHAT IS KNOWN ALREADY: RPL not only causes the women affected and their partners considerable emotional distress; it also has an impact on the couples' relationships and the way they relate to their families and friends. Studies suggest that women have a greater need than their male partners to talk about their losses and that these differences may lead to dissatisfaction and cause relational tension. In addition, men often assume a 'mainstay' role, supporting their partners and displaying fortitude in the face of distress. As yet, however, little research has been conducted so far on the question of what the members of couples with RPL expect from one another and from their families and friends. STUDY DESIGN SIZE DURATION: The study sample consisted of 147 couples and 17 women with at least 2 miscarriages attending the special unit for RPL at the University Women's Hospital in Heidelberg (Germany) for the first time between September 2018 and October 2020 (response rate: 82.7%). The patients were asked to participate in this combined qualitative and questionnaire study. PARTICIPANTS/MATERIALS SETTING METHODS: In order to explore the wishes and needs of those affected in more detail, the free text responses obtained were examined in this study by using qualitative content analysis. Categories and subcategories were created inductively to summarize and systematize content. MAIN RESULTS AND THE ROLE OF CHANCE: Patients affected by RPL want their partners and their families and friends to deal with the topic openly and empathically. In the partnership itself, acceptance of individual grieving modes and sharing a common goal are important factors. Men, in particular, want their partners to be optimistic in facing up to the situation. Regarding communication with family and friends, it transpired that 'good advice', playing the matter down, inquiries about family planning, pity and special treatment are explicitly not appreciated. LIMITATIONS REASONS FOR CAUTION: The sample was a convenience sample, so self-selection effects cannot be excluded. In addition, the level of education in the sample was above average. Accordingly, the sample cannot be regarded as representative. The results of the content analysis are based on the respondents' written answers to open-ended questions in the questionnaire. Unlike qualitative interview studies, further questioning was not possible in the case of ambiguities or to request more details. WIDER IMPLICATIONS OF THE FINDINGS: Frank and sincere communication about miscarriages and about one's own emotions and needs should be promoted both in the partnership and among family members and friends in order to strengthen the potential of social support as a resource. Open communication about the different needs of both partners is necessary to create mutual understanding. The results show the importance not only of empathy and consideration for the couples concerned but also their desire not to be pitied. Striking a fine balance between fellow-feeling and pity may also lead to tension, and this potential dilemma should be addressed in psychosocial counselling. Overall, the study contributes to a better understanding of what couples want from their families and friends when they are attempting to come to terms with RPL and highlights potential challenges in the interaction between affected couples and their families and friends. STUDY FUNDING/COMPETING INTERESTS: No funding was received for this study. None of the authors declared any conflicts of interest. TRIAL REGISTRATION NUMBER: DRKS00014965.

Human AZFb deletions cause distinct testicular pathologies depending on their extensions in Yq11 and the Y haplogroup: new cases and review of literature.

Genomic AZFb deletions in Yq11 coined "classical" (i.e. length of Y DNA deletion: 6.23 Mb) are associated with meiotic arrest (MA) of patient spermatogenesis, i.e., absence of any postmeiotic germ cells. These AZFb deletions are caused by non-allelic homologous recombination (NAHR) events between identical sequence blocks located in the proximal arm of the P5 palindrome and within P1.2, a 92 kb long sequence block located in the P1 palindrome structure of AZFc in Yq11. This large genomic Y region includes deletion of 6 protein encoding Y genes, EIFA1Y, HSFY, PRY, RBMY1, RPS4Y, SMCY. Additionally, one copy of CDY2 and XKRY located in the proximal P5 palindrome and one copy of BPY1, two copies of DAZ located in the P2 palindrome, and one copy of CDY1 located proximal to P1.2 are included within this AZFb microdeletion. It overlaps thus distally along 2.3 Mb with the proximal part of the genomic AZFc deletion. However, AZFb deletions have been also reported with distinct break sites in the proximal and/or distal AZFb breakpoint intervals on the Y chromosome of infertile men. These so called "non-classical" AZFb deletions are associated with variable testicular pathologies, including meiotic arrest, cryptozoospermia, severe oligozoospermia, or oligoasthenoteratozoospermia (OAT syndrome), respectively. This raised the question whether there are any specific length(s) of the AZFb deletion interval along Yq11 required to cause meiotic arrest of the patient's spermatogenesis, respectively, whether there is any single AZFb Y gene deletion also able to cause this "classical" AZFb testicular pathology? Review of the literature and more cases with "classical" and "non-classical" AZFb deletions analysed in our lab since the last 20 years suggests that the composition of the genomic Y sequence in AZFb is variable in men with distinct Y haplogroups especially in the distal AZFb region overlapping with the proximal AZFc deletion interval and that its extension can be "polymorphic" in the P3 palindrome. That means this AZFb subinterval can be rearranged or deleted also on the Y chromosome of fertile men. Any AZFb deletion observed in infertile men with azoospermia should therefore be confirmed as "de novo" mutation event, i.e., not present on the Y chromosome of the patient's father or fertile brother before it is considered as causative agent for man's infertility. Moreover, its molecular length in Yq11 should be comparable to that of the "classical" AZFb deletion, before meiotic arrest is prognosed as the patient's testicular pathology.

Gonadoblastoma Y locus genes expressed in germ cells of individuals with dysgenetic gonads and a Y chromosome in their karyotypes include DDX3Y and TSPY.

STUDY QUESTION: Which Y genes mapped to the 'Gonadoblastoma Y (GBY)' locus on human Y chromosome are expressed in germ cells of individuals with some Differences of Sexual Development (DSD) and a Y chromosome in their karyotype (DSD-XY groups)? SUMMARY ANSWER: The GBY candidate genes DDX3Y and TSPY are expressed in the germ cells of DSD-XY patients from distinct etiologies: patients with mixed gonadal dysgenesis (MGD) and sex chromosome mosaics (45,X0/46,XY; 46,XX/46,XY); patients with complete androgen insensitivity (CAIS), patients with complete gonadal dysgenesis (CGD; e.g. Swyer syndrome). WHAT IS KNOWN ALREADY: A GBY locus was proposed to be present on the human Y chromosome because only DSD patients with a Y chromosome in their karyotype have a high-although variable-risk (up to 55%) for germ cell tumour development. GBY was mapped to the proximal part of the short and long Y arm. TSPY located in the proximal part of the short Y arm (Yp11.1) was found to be a strong GBY candidate gene. It is expressed in the germ cells of DSD-XY patients with distinct etiologies but also in foetal and pre-meiotic male spermatogonia. However, the GBY region extends to proximal Yq11 and therefore includes probably more than one candidate gene. STUDY DESIGN, SIZE, DURATION: Protein expression of the putative GBY candidate gene in proximal Yq11, DDX3Y, is compared with that of TSPY in serial gonadal tissue sections of 40 DSD-XY individuals from the three DSD patient groups (MGD, Complete Androgen Insensitivity Syndrome [CAIS], CGD) with and without displaying malignancy. Expression of OCT3/4 in the same tissue samples marks the rate of pluripotent germ cells. PARTICIPANTS/MATERIALS, SETTING, METHOD: A total of 145 DSD individuals were analysed for the Y chromosome to select the DSD-XY subgroup. PCR multiplex assays with Y gene specific marker set score for putative microdeletions in GBY Locus. Immunohistochemical experiments with specific antisera mark expression of the GBY candidate proteins, DDX3Y, TSPY, in serial sections of the gonadal tissue samples; OCT3/4 expression analyses in parallel reveal the pluripotent germ cell fraction. MAIN RESULTS AND THE ROLE OF CHANCE: Similar DDX3Y and TSPY protein expression patterns were found in the germ cells of DSD-XY patients from each subgroup, independent of age. In CAIS patients OCT3/4 expression was often found only in a fraction of these germ cells. This suggest that GBY candidate proteins are also expressed in the non-malignant germ cells of DSD-XY individuals like in male spermatogonia. LIMITATIONS, REASONS FOR CAUTION: Variation of the expression profiles of GBY candidate genes in the germ cells of some DSD-XY individuals suggests distinct transcriptional and translational control mechanisms which are functioning during expression of these Y genes in the DSD-XY germ cells. Their proposed GBY tumour susceptibility function to transform these germ cells to pre-malignant GB/Germ Cell Neoplasia in Situ (GB/GCNIS) cells seems therefore to be limited and depending on their state of pluripotency. WIDER IMPLICATIONS OF THE FINDINGS: These experimental findings are of general importance for each individual identified in the clinic with DSD and a Y chromosome in the karyotype. To judge their risk of germ cell tumour development, OCT3/4 expression analyses on their gonadal tissue section is mandatory to reveal the fraction of germ cells still being pluripotent. Comparative expression analysis of the GBY candidate genes can be helpful to reveal the fraction of germ cells with genetically still activated Y chromosomes contributing to further development of malignancy if at high expression level. STUDY FUNDING/COMPETING INTEREST(S): This research project was supported by a grant (01GM0627) from the BMBF (Bundesministerium für Bildung und Forschung), Germany to P.H.V. and B.B. The authors have no competing interests.

Psychometric characteristics of the FertiQoL questionnaire in a German sample of infertile individuals and couples.

BACKGROUND: FertiQoL is a questionnaire internationally developed to measure fertility-specific quality of life. It has been validated with infertile populations in many countries and used in several studies focusing on the psychosocial consequences of infertility in Europe, Asia, and North America. METHODS: Over a period of two years, 596 infertile women and men took part in the study conducted at three German fertility clinics. Psychometric properties of FertiQoL were tested by performing confirmatory factor analyses, calculating average variance extracted values, reliability and correlation coefficients. Hierarchical regression analyses were conducted to determine the relations between FertiQoL subscales and both sociodemographic and medical variables. Individual and cross-partner effects were tested for. RESULTS: The confirmatory factor analyses conducted on our FertiQoL data supported the original four-factor solution for both women and men but, resulted in some unsatisfactory indices. Family and friends' support items loaded weakly on the Social subscale of FertiQoL (.27 and .34 in women, .32 and .19 in men). The Emotional and Mind/Body subscales revealed a strong intercorrelation (r = .77, p < .001 in women, r = .74, p < .001 in men). Women scored lower than men on the Emotional and Mind/Body subscales only, and they reported better fertility-specific relational QoL. In women, the perceived cause of infertility and already mothering a child related significantly to individual FertiQoL scores, while in men, age, educational level, and the duration of their wish for a child had an impact on the FertiQoL subscales (all p < .05). The men's educational level, the women's educational level, and the subjective perceived medical cause of fertility problems exerted cross-partner effects on QoL (all p < .05). CONCLUSIONS: Our study results represent a contribution both to research and clinical practice. The findings suggest the importance of considering the personal experience of infertility in different cultural and gender specific settings and that the strong connections between the emotional, physical, and cognitive aspects of an individual's fertility-specific quality of life should be regarded as a more coherent system. TRIAL REGISTRATION: DRKS: DRKS00014707 . Registered 1 May 2018 (retrospectively registered).

Fertility preservation: ovarian response to freeze oocytes is not affected by different malignant diseases-an analysis of 992 stimulations.

PURPOSE: To study if ovarian response is affected by the type of disease if fertility preservation is required. METHODS: A registry of the trinational fertility preservation network FertiPROTEKT including 992 patients aged 18-40 years undergoing ovarian stimulation and follicle aspiration for fertility preservation from 1/2007 until 3/2016 was analysed. The number of collected oocytes, days of stimulation, total gonadotropin dosage and gonadotropin dosage per day were evaluated. RESULTS: Total oocyte number was negatively correlated with increasing age (r = 0.237, p < 0.0001). Oocyte numbers were in women < 26 years 15.4 ± 8.8, 26-30 years 13.1 ± 8.5, 31-35 years 12.2 ± 7.7 and 36-40 years 9.9 ± 8.0. Age-adjusted oocyte numbers were not different in women with Hodgkin's lymphoma (12.6 ± 8.8), non-Hodgkin's lymphoma (12.4 ± 8.2), leukaemia (11.7 ± 8.2), sarcoma (11.8 ± 8.2), cerebral cancer (16.5 ± 8.1), gastrointestinal cancer (13.2 ± 8.1) gynaecological cancer (10.8 ± 8.2) and other types of malignancies (15.8 ± 8.1) apart from ovarian cancer with lower oocyte yield (7.3 ± 8.3, p < 0.001) compared to women with breast cancer (13.3 ± 8.8). The total gonadotropin dose used for stimulation was only elevated in Hodgkin's and non-Hodgkin's lymphoma compared to women with breast cancer (p < 0.05). Oocyte yield was lower in women with versus without ovarian cancer (p < 0.0001). CONCLUSIONS: As ovarian response is not affected by the type of cancer, ovarian stimulation can be performed with the same oocyte yield in different malignant diseases. However, oocyte yield is reduced if ovarian surgery is required and in older women.

Serum periostin levels in early in pregnancy are significantly altered in women with miscarriage.

BACKGROUND: Miscarriage is a common complication in pregnancy and there is still a lack of biomarkers usable in asymptomatic patients before the event occurs. Periostin (PER), whose levels rise particularly during injury or inflammation, has been shown to play an important local role in implantation and early embryonic development. As PER has been described as a biomarker in various medical conditions we intended to evaluate if changes in PER serum levels may help to identify women at risk for spontaneous abortion in the first trimester. METHODS: Women between 18 and 42 years without confounding comorbidities who conceived by IVF/ICSI and ovarian hyperstimulation were analysed in the study after informed consent. Maternal serum samples from 41 patients were assessed at the time of pregnancy testing (PT) and the following first ultrasound checkup (US). Patients were subsequently divided in two groups: (1) patients with subsequent miscarriage in the first trimester (n = 18) and (2) patients with ongoing pregnancy (n = 23), allowing for statistical analysis and investigating the change of PER levels per individual. PER levels were measured using enzyme-linked immunosorbent assay. Statistical analysis was performed using the Fisher exact and Student's t test. p ≤ 0.05 was considered to be significant. RESULTS: There was no significant difference concerning possible confounders between the two groups. We did not find any significant difference in PER levels at the time point of PT or US. By investigating the interindividual changes of PER between the two time points however, we observed that patients with a following miscarriage showed increasing levels of PER at the time point of PT compared to US in contrast to patients with an ongoing pregnancy who demonstrated a decrease in PER levels. These alterations were significant in the absolute as well as in the relative comparison. CONCLUSION: The relative expression of PER between PT and US is significantly altered in asymptomatic women with subsequent miscarriage compared to women with ongoing pregnancy. Therefore systemic PER levels might represent a potential promising biomarker for the assessment of pregnancy outcome. TRIAL REGISTRATION: Not applicable.

Higher prevalence of colonization with Gardnerella vaginalis and gram-negative anaerobes in patients with recurrent miscarriage and elevated peripheral natural killer cells.

The role of vaginal infections in recurrent miscarriage (RM) is discussed controversially and screening is not recommended in international guidelines. Peripheral and uterine NK cells (pNK, uNK) play an important role in the establishment of a healthy pregnancy and are targets of immune diagnostics in RM patients. The aim of this study was to analyze the composition of the vaginal microbiota in RM patients and to correlate the findings to clinical characteristics as well as NK cell parameters. In total, n=243 RM patients with ≥3 consecutive miscarriages were recruited between 11/2011 and 03/2016. Vaginal swabs were analyzed by microbiological culture. Further, a cervical swab was taken in n=187 patients and the presence of Chlamydia trachomatis was evaluated by a molecular assay. Peripheral blood levels of CD45+CD3-CD56+CD16+ pNK (determined by four-color fluorescence flow cytometry) and CD56+ uNK (uterine biopsy, determined by immunohistochemistry) were analyzed. The prevalence of Gardnerella vaginalis colonization in RM patients was 19.0%, gram-negative anaerobes 20.5%, Candida species 7.9%, group B Streptococcus 11.0% and Enterobacteriaceae 14.8%. Commensal lactobacilli were absent in 14.5% of the women. Chlamydia trachomatis was detected in n=1 case (0.53%). The prevalence of Gardnerella vaginalis and gram-negative anaerobes in RM patients with elevated pNK (>280/µl, n=69) was significantly higher (p=0.012, p=0.04) compared to patients with normal pNK (n=174). In conclusion, RM patients with elevated pNK suffer more often from colonization by Gardnerella vaginalis and gram-negative anaerobes. This might indicate an association between the vaginal microbiota, local inflammation, changes in immune parameters and miscarriage.

Two-year development of children conceived by IVM: a prospective controlled single-blinded study.

STUDY QUESTION: Is there a difference in mental development of children conceived by IVM in comparison to IVF or ICSI, independently, at the age of 2 years? SUMMARY ANSWER: No differences could be found in mental development of IVM children compared to IVF and IVM children compared to ICSI as well. WHAT IS KNOWN ALREADY: Only few retrospective or non-controlled studies addressed the health of IVM children and did not show a negative impact of the IVM procedure. STUDY DESIGN, SIZE, DURATION: Prospective controlled single-blinded study including 63 pregnancies (21 per IVM, IVF and ICSI groups) with 70 children expected. Examinations of 62 embryos at first trimester screening, of 57 fetuses at 21st week of pregnancy, of 60 children at birth and of 37 children at their second birthday were performed during the study period from January 2009 until October 2016. Bayley score at the age of 2 was the primary outcome parameter. Data of 40 children after spontaneous conception from a previous prospective unrelated study were further used as control at 2 years examination and compared to the pooled ART group (IVM, IVF and ICSI). PARTICIPANTS/MATERIALS, SETTING, METHODS: Twenty-one IVM pregnancies achieved in the study period were included. For each of them, the following IVF- and ICSI pregnancies were recruited as controls. Ultrasound examinations during pregnancy, examinations of newborns and of children around their second birthday were done by blinded prenatal specialists, pediatricians and neuropediatricians, respectively. MAIN RESULTS AND THE ROLE OF CHANCE: Children conceived after IVM did not show differences during embryonic development, at birth nor in their neuropediatric development at the age of 2 compared to their counterparts after IVF and after ICSI (Bayley score 91.3 ± 21.0 for IVM, 96.8 ± 13.2 for IVF and 103.9 ± 13.1 for ICSI) and of the pooled ART group compared to children after spontaneous conception (96.6 ± 16.4 ART and 103.2 ± 9.4 spontaneous conception). When analyzing singleton pregnancies only, again no differences during pregnancy, at birth and at their 2-year evaluation were detected between IVM versus IVF and IVM versus ICSI. LIMITATIONS, REASONS FOR CAUTION: Due to the small sample size data must be interpreted with caution. To allow a confirmative answer that there are no health risks for children conceived by IVM, large multicenter cohort or registry-based studies are urgently needed. WIDER IMPLICATIONS OF THE FINDINGS: The study adds further information to previous uncontrolled or retrospective studies, which were unable to detect risks for the health of IVM children. STUDY FUNDING/COMPETING INTEREST(S): The study was supported by the 'Deutsche Forschungsgemeinschaft' (DFG): STR 387/4-1. G.R. receives royalties from Pearson Assessment Germany (editor fee for Bayley-III). The other authors have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: Not applicable.

Infertile Partners' Coping Strategies Are Interrelated - Implications for Targeted Psychological Counseling.

Background Infertility patients often have high stress levels which, in some cases, represent a risk of developing depression or anxiety. The SCREENIVF questionnaire is a validated tool to evaluate such risks. Some coping strategies have been shown to be correlated with infertile couples' levels of stress. Determining which strategies are correlated with higher levels of risk for depression or anxiety could be useful to offer targeted psychological counseling to reduce the risk of depression or anxiety. Materials and Methods A total of 296 women and men who attended the Fertility Center at Heidelberg University Hospital completed the SCREENIVF questionnaire and the COMPI coping scales. Data were analyzed first on an individual basis and focused on the couple, using the Actor Partner Interdependence Model. Results On an individual level, active avoidance coping was positively correlated with a higher risk of depression or anxiety in women, while meaning-based coping was negatively correlated with risk in men. When the results of couples were viewed together, women and men using active avoidance coping exhibited higher risk scores as individuals (actor effect), as did their partners (partner effect). Women who used meaning-based coping had positive actor and partner effects. Women using active-confronting coping had a negative partner effect (higher risk score for men). Conclusions These findings indicate that some coping strategies may have a protective effect while others may increase the risk of emotional maladjustment in infertile couples. Further analysis of coping strategies could help to identify new counseling approaches for infertile patients.

Natural conception rates in subfertile couples following fertility awareness training.

PURPOSE: To analyze cumulative pregnancy rates of subfertile couples after fertility awareness training. METHODS: A prospective observational cohort study followed 187 subfertile women, who had received training in self-observation of the fertile phase of the menstrual cycle with the Sensiplan method, for 8 months. The women, aged 21-47 years, had attempted to become pregnant for 3.5 years on average (range 1-8 years) before study entry. Amenorrhea, known tubal occlusion and severe male factor had been excluded. An additional seven women, who had initially been recruited, became pregnant during the cycle immediately prior to Sensiplan training: this is taken to be the spontaneous pregnancy rate per cycle in the cohort in the absence of fertility awareness training. RESULTS: The cumulative pregnancy rate of subfertile couples after fertility awareness training was 38% (95% CI 27-49%; 58 pregnancies) after eight observation months, which is significantly higher than the estimated basic pregnancy rate of 21.6% in untrained couples in the same cohort. For couples who had been seeking to become pregnant for 1-2 years, the pregnancy rate increased to 56% after 8 months. A female age above 35 (cumulative pregnancy rate 25%, p = 0.06), couples who had attempted to become pregnant for more than 2 years (cumulative pregnancy rate 17%, p < 0.01), all significantly reduce the chances of conceiving naturally at some point. CONCLUSIONS: Training women to identify their fertile window in the menstrual cycle seems to be a reasonable first-line therapy in the management of subfertility.

The "killer cell story" in recurrent miscarriage: Association between activated peripheral lymphocytes and uterine natural killer cells.

Peripheral and uterine NK cells (pNK, uNK) can be distinguished according to their receptor expression. Recent studies indicate an association of elevated pNK and uNK with recurrent miscarriage (RM). This study aimed to analyze pNK and uNK in patients with RM and healthy controls. Out of n=590 RM patients screened according to a standard diagnostic protocol, n=268 couples with ≥3 consecutive RM were identified. Subgroups consisted of n=151 primary RM (pRM), n=85 secondary RM (sRM), n=32 tertiary RM (tRM) and n=42 healthy controls. Finally, n=147 idiopathic RM (iRM) and n=121 non-iRM patients were identified. Peripheral blood levels of CD45+CD3-CD56+CD16+ NK cells were determined in non-pregnant patients and controls in the mid-luteal phase by FACS. In n=129 RM patients a uterine biopsy was taken to evaluate CD56+ NK cells by immunohistochemistry. PRM showed higher absolute pNK than sRM (median/µl (Q1;Q3): 234 (147;306) vs 176 (128;245), p=0.02). Further a trend towards higher pNK percentages in pRM was detected. UNK numbers did not differ between RM subgroups and did not correlate with pNK. However, the rate of highly elevated uNK was increased in iRM compared to non-iRM patients (p=0.04). Further, higher numbers of CD45+CD3-DR+ (p<0.01) and CD45+CD3+CD8+DR+ (p=0.04) peripheral lymphocytes were associated with higher uNK numbers. In conclusion, elevated pNK were present in pRM patients. Although pNK and uNK numbers did not correlate, the association between high CD45+CD3-DR+ and CD45+CD3+CD8+DR+ peripheral lymphocytes and uNK might indicate that activated NK, B and T cells provide cytokines for the differentiation of uNK.

Effects of medical causes, role concepts and treatment stages on quality of life in involuntary childless men.

Goal of this study was to investigate differences in quality of life in men contingent upon various fertility treatment stages, infertility causes and adoption of roles. A quantitative study with n = 115 men in three German fertility centres was devised. Participants completed a standardised, fertility-specific questionnaire devised for men (TLMK), sociodemographic and role items. Men having experienced severe medical conditions, for example cancer, reported significant higher quality of life compared to men with other infertility reasons [F(1,56) = 12.77, P = 0.001]. Furthermore, allocating participants into distinctive groups by means of kind and duration of treatment revealed significant group differences [F(2,111) = 4.94, P = 0.009], with quality of life decreasing with the use of more invasive fertility methods. A higher satisfaction with life was also stated by men adopting many tasks in the treatment process. The high quality of life displayed by men having experienced severe medical conditions contains valuable and far-reaching information about possible resilience factors that need to be researched more in detail. The finding of decreasing quality of life in men with the use of more invasive methods in treatment applies for increased psychosocial services in fertility clinics.

Patients with idiopathic recurrent miscarriage show higher levels of DR+ activated T-cells that are less responsive to mitogens.

In 50% of recurrent miscarriages (RM) the cause remains unknown and standardized immunological diagnosis and treatment of idiopathic RM (iRM) is yet not established. In this prospective case-control study, out of 220 RM patients screened, 97 iRM patients were identified and compared to 26 healthy controls without a previous pregnancy or blood transfusion in order to identify deregulated immunological parameters. Blood levels of lymphocyte subpopulations, cytokines and neopterin were determined by FACS, ELISA, and Luminex technique. Lymphocyte function was studied by in-vitro lympocyte proliferation tests. As compared to controls, patients had significantly higher proportions of activated CD3+DR+, CD4+DR+ and CD8+DR+ lymphocytes, elevated levels of neopterin and a lower in-vitro proliferation of lymphocytes (all p<0.05). Within the iRM patients higher proportions of CD3+DR+ T-lymphocytes correlated with higher proportions and absolute numbers of CD4+DR+ and CD8+DR+ T-lymphocytes and lower CD16+CD56+ NK-cells. Further, it was associated with lower absolute numbers of CD19+ B-lymphocytes, CD3+CD25+ T-lymphocytes and CD45+ total lymphocytes (all p<0.05). In addition we found decreased in-vitro lymphocyte proliferation in iRM patients with high CD3+DR+ T-lymphocytes (p<0.05). In summary patients with iRM showed increased activated T-cells that are less responsive to mitogens in-vitro. The inverse relationship of increased DR but decreased CD25 expression on CD3+ T-cells and the decreased in-vitro proliferation characterize an immunological disorder with similarities to T-cell exhaustion in patients with HIV and cancer. These abnormalities potentially contribute to the pathogenesis of iRM and might be a target for future immunomodulatory therapies.

Anti-Mullerian-Hormone during pregnancy and peripartum using the new Beckman Coulter AMH Gen II Assay.

BACKGROUND: AMH levels determined by the conventional AMH assay declined during pregnancy and postpartum. A new Beckman Coulter AMH Gen II assay removes the potentially assay-interfering complement which is activated in pregnancy. The aim of this study was to evaluate if the decline of AMH levels in the serum of pregnant women during the course of pregnancy and peripartum was assay-dependent and thus artificial. METHODS: In this cross-sectional study prepartal blood samples were collected from 62 patients (median age 30.6 years [interquartile range: 25.6 - 34.5]) in the third trimester of pregnancy and again 1-4 days after delivery between 2011 and 2012. In another cohort of 11 patients (median age 34.1 years [interquartile range: 32.6 - 37.8]) blood samples were taken in different trimesters of pregnancy between 1995 and 2001. The conventional and the modified AMH assay were performed in the same patient serum samples. We used the conventional and the modified AMH-Gen-II ELISA (Beckman Coulter, Immunotech, Webster, USA) for the assessment of AMH levels. The Wilcoxon signed rank test was used for determining differences between AMH levels pre- and postpartum. The method of Bland and Altman was applied for analyzing the agreement of both methods for determining AMH levels. RESULTS: AMH values peripartum were lower than those expected in fertile non-pregnant women of comparable age. An overall mean difference of 0.44 ng/ml was observed between the conventional and the modified assay. Measurements with the modified assay showed a significant decline of postpartal levels compared with prepartal levels which is consistent with values obtained using the conventional assay (both p < 0.00001). Compared to the longitudinal measurements of AMH levels determined using the conventional assay, AMH levels obtained using the modified assay suggest a steeper decline of values during the course of pregnancy. CONCLUSION: By comparing the conventional assay for AMH determination with the modified assay the present study confirmed that AMH levels decline during the course of pregnancy and early after delivery.

A multi-centre phase 3 study comparing efficacy and safety of Bemfola(®) versus Gonal-f(®) in women undergoing ovarian stimulation for IVF.

Bemfola (follitropin alfa) (Finox AG, Switzerland), a new recombinant FSH, has a comparable pharmacological profile to that of Gonal-f (Merck Serono, Germany), the current standard for ovarian stimulation. A randomized, multi-centre, Phase 3 study in women undergoing IVF or intracytoplasmic sperm injection (n = 372) showed Bemfola yielding similar efficacy and safety profiles to Gonal-f. Women aged 20-38 years of age were randomized 2:1 to receive a single, daily, subcutaneous 150 IU dose of either Bemfola or Gonal-f. This study tested equivalence in the number of retrieved oocytes using a pre-determined clinical equivalence margin of ±2.9 oocytes. Compared with Gonal-f, Bemfola treatment resulted in a statistically equivalent number of retrieved oocytes (Bemfola 10.8 ± 5.11 versus Gonal-f 10.6 ± 6.06, mean difference: 0.27 oocytes, 95% confidence interval: -1.34, 1.32) as well as a similar clinical pregnancy rate per embryo transfer in first and second cycles (Bemfola: 40.2% and 38.5%, respectively; Gonal-f: 48.2% and 27.8%, respectively). No difference in severe ovarian hyperstimulation syndrome was observed between treatment groups (Bemfola: 0.8%; Gonal-f: 0.8%). This study demonstrates similar clinical efficacy and safety profiles between Bemfola and Gonal-f, and suggests that Bemfola can be an appropriate alternative in ovarian stimulation protocols.

Immune profiling in patients with recurrent miscarriage.

The central role of the maternal immune system for successful and disturbed pregnancies such as recurrent miscarriage (RM) is apparent. Recent studies have increased understanding of the complex interaction of the different immunological players and the adaptation of the maternal immune system to the semi-allogeneic embryo. There is growing evidence for immunological abnormalities in RM patients, including autoimmune and allogeneic factors. However, the question remains unsolved whether these changes represent the cause or the consequence of RM. As in half of the RM patients the underlying mechanism remains unknown, further diagnostic methods are urgently needed. Within this review we summarize (recent) literature on the immunological diagnosis in RM patients to find out current trends and to identify potential targets of therapy. As the exact mechanisms of feto-maternal tolerance have not yet been determined we suggest that the immunological diagnosis should be implemented only in well-designed clinical trials in specialized centers to establish a standardized immunological work-up in RM patients.

Change of anti-Mullerian-hormone levels during follicular phase in PCOS patients.

Anti-Mullerian-hormone (AMH) does not seem to fluctuate significantly during the menstrual cycle in healthy women. However, little is known about cycle fluctuations of AMH levels in patients with polycystic ovarian syndrome (PCOS). The purpose of this study was to examine AMH fluctuations during the follicular phase in PCOS patients receiving antiestrogens or recombinant follicle-stimulating hormone (FSH). About 40 PCOS patients diagnosed according to Rotterdam ESHRE/ASRM-Sponsored PCOS Consensus Workshop Group 2003 and 19 controls were prospectively enrolled. PCOS patients received either antiestrogens or recombinant FSH for monoovulation induction and controls received antiestrogens. AMH levels were determined (1) between the 2nd and the 5th day of follicular phase and (2) when a single large dominant follicle ≥18 mm had appeared. Our study shows that AMH levels do not change during follicular development in controls as well as in PCOS patients with AMH levels < 5 ng/ml, irrespective of antiestrogen or FSH therapy. However, in PCOS patients with AMH levels ≥5 ng/ml, AMH declines significantly during follicular development (p < 0.01). We conclude that AMH levels should be determined in the early follicular phase in PCOS patients without the influence of antiestrogens or exogenous FSH, because these interventions may lower AMH values in patients with high levels.

Male immunity to the chlamydial 60 kDa heat shock protein (HSP 60) - associated with semen quality?

The role of Chlamydia trachomatis for male infertility is a matter of constant debate. It is assumed that in its persistent form this pathogen may produce high levels of 60 kD heat shock protein (Chlam HSP60). Cross-reactivity between epitopes of the bacterial and human HSPs, involved in many steps of the reproductive process, might induce an autoimmune response with potential impairment of semen quality and sperm fertilising capacity. This prospective study included asymptomatic males of a total of 128 unselected subfertile couples (median duration of infertility 3 years) to determine the clinical relevance of male immunity to Chlam HSP60 during infertility investigation. After medical history and clinical examination of both partners, serum antibodies (Ab) to Chlam HSP60 were determined. Same day semen quality evaluation included microscopical standard sperm analysis, determination of seminal white blood cells (WBC) and of antisperm Ab (ASA) of the Ig G- and Ig-A class (mixed antiglobulin reaction, MAR), microbial screening and examination of sperm functional capacity. Sperm/mucus interaction was tested in vitro and in vivo. Simultaneously, patients' female partners were tested for Chlam HSP60 Ab and results were compared with a standard serology evaluation for antichlamydial IgG Ab. The presence of ChlamHSP60 Ab (positive in 24% of males) was not significantly associated with semen quality, seminal WBC and antisperm AB of the IgG- or Ig A-class, the outcome of the microbial screening nor with sperm functional capacity and results of sperm/mucus interaction testing in vitro and in vivo. Chlam HSP60 Ab were significantly more frequent in female partners of Chlam HSP60 Ab-positive men, and results correlated with the outcome of standard chlamydial serology evaluation. In conclusion, when serum Chlam HSP60 Ab are used as marker, male immunity to the chlamydial 60 kD heat shock protein is not associated with semen quality, sperm functional capacity and other clinically relevant parameters of male fertility.