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Category Archives: Human Reproduction
ART culture conditions change the probability of mouse embryo gestation through defined cellular and molecular responses
STUDY QUESTION
Do different human ART culture protocols prepare embryos differently for post-implantation development?
SUMMARY ANSWER
The type of ART culture protocol results in distinct cellular and molecular phenotypes in vitro at the blastocyst stage as well as subsequently during in vivo development.
WHAT IS KNOWN ALREADY
It has been reported that ART culture medium affects human development as measured by gestation rates and birthweights. However, due to individual variation across ART patients, it is not possible as yet to pinpoint a cause–effect relationship between choice of culture medium and developmental outcome.
STUDY DESIGN, SIZE, DURATION
In a prospective study, 13 human ART culture protocols were compared two at a time against in vivo and in vitro controls. Superovulated mouse oocytes were fertilized in vivo using outbred and inbred mating schemes. Zygotes were cultured in medium or in the oviduct and scored for developmental parameters 96h later. Blastocysts were either analyzed or transferred into fosters to measure implantation rates and fetal development. In total, 5735 fertilized mouse oocytes, 1732 blastocysts, 605 fetuses and 178 newborns were examined during the course of the study (December 2010–December 2011).
PARTICIPANTS/MATERIALS, SETTING, METHODS
Mice of the B6C3F1, C57Bl/6 and CD1 strains were used as oocyte donors, sperm donors and recipients for embryo transfer, respectively. In vivo fertilized B6C3F1 oocytes were allowed to cleave in 13 human ART culture protocols compared with mouse oviduct and optimized mouse medium (KSOM(aa)). Cell lineage composition of resultant blastocysts was analyzed by immunostaining and confocal microscopy (trophectoderm, Cdx2; primitive ectoderm, Nanog; primitive endoderm, Sox17), global gene expression by microarray analysis, and rates of development to midgestation and to term.
MAIN RESULTS AND THE ROLE OF CHANCE
Mouse zygotes show profound variation in blastocyst (49.9–91.9%) and fetal (15.7–62.0%) development rates across the 13 ART culture protocols tested (R2= 0.337). Two opposite protocols, human tubal fluid/multiblast (high fetal rate) and ISM1/ISM2 (low fetal rate), were analyzed in depth using outbred and inbred fertilization schemes. Resultant blastocysts show imbalances of cell lineage composition; culture medium-specific deviation of gene expression (38 genes, ≥4-fold) compared with the in vivo pattern; and produce different litter sizes (P ≤ 0.0076) after transfer into fosters. Confounding effects of subfertility, life style and genetic heterogeneity are reduced to a minimum in the mouse model compared with ART patients.
LIMITATIONS, REASONS FOR CAUTION
This is an animal model study. Mouse embryo responses to human ART media are not transferable 1-to-1 to human development due to structural and physiologic differences between oocytes of the two species.
WIDER IMPLICATIONS OF THE FINDINGS
Our data promote awareness that human ART culture media affect embryo development. Effects reported here in the mouse may apply also in human, because no ART medium presently available on the market has been optimized for human embryo development. The mouse embryo assay (MEA), which requires ART media to support at least 80% blastocyst formation, is in need of reform and should be extended to include post-implantation development.
STUDY FUNDING/COMPETING INTEREST(S)
This work was supported by the Deutsche Forschungsgemeinschaft (BO 2540/4-1 to M.B. and SCHL 394/9 to S.S.) and by the Nederlandse Organisatie voor Wetenschappelijk Onderzoek (no. 63-258 to S.L.G.). No competing interests.
TRIAL REGISTRATION NUMBER
Not applicable.
Posted in Human Reproduction
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Expression of adhesion, attachment and invasion markers in eutopic and ectopic endometrium: a link to the aetiology of endometriosis
BACKGROUND
Cell properties, such as attachment, adhesion and invasion, are important for the normal function of the endometrium. However, it is believed that the same properties may also be involved in the development of gynaecological diseases, such as endometriosis. Endometrial cells, shed by retrograde menstruation, may have an aberrant expression of molecules involved in these functions, leading to endometriosis. Therefore, the aim of this study was to investigate the expression of proteins involved in adhesion, attachment and invasion in eutopic and ectopic endometrium.
METHODS
Endometrial biopsy specimens were collected from healthy volunteers (controls: proliferative phase, n = 10; secretory phase, n = 15) and from endometriosis patients (proliferative phase: n = 9, secretory phase: n = 10). Biopsy specimens from endometriomas were also collected (proliferative phase: n = 9, secretory phase: n = 10). Expression of apolipoprotein E (ApoE), integrin β-2 (ITGB2), integrin β-7 (ITGB7), Laminin -1 (LAMC1), CD24 molecule (CD24) and junctional adhesion molecule-1 (JAM-1) was evaluated with real-time reverse transcriptase polymerase chain reaction and immunohistochemistry.
RESULTS
The endometrium from controls and women with endometriosis expressed ApoE, ITGB2, ITGB7, LAMC1, CD24 and JAM-1. Gene expression of ApoE and JAM-1 was decreased in both proliferative and secretory phase in the endometrium from women with endometriosis compared with control endometrium. Also, mRNA expression of LAMC1 was reduced in the endometrium from endometriosis patients compared with controls in the proliferative phase. An altered gene expression of CD24 was seen between the endometrium from endometriosis patients and endometriomas in the secretory phase. The ITGB2 protein expression was altered in epithelia cells between the endometrium from healthy volunteers and endometriosis patients in the secretory phase.
CONCLUSIONS
We have shown differential expression of adhesion, attachment and invasion proteins in proliferative and secretory endometrium from controls and endometriosis patients and in endometriomas. This study suggests that molecules with these properties may have a role in the anchoring of endometrial cells at ectopic sites, thus initiating the development of endometriosis.
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Psychometric properties of the Iranian version of modified polycystic ovary syndrome health-related quality-of-life questionnaire
STUDY QUESTION
Can the modified polycystic ovary syndrome health-related quality-of-life questionnaire (MPCOSQ) be validated in Iranian patients with polycystic ovary syndrome (PCOS) to provide an instrument for future outcome studies in these patients?
SUMMARY ANSWER
We show initial reliability and validity of the Iranian version of the MPCOSQ as a specific instrument to measure health-related quality of life (QOL) in patients with PCOS in Iran.
WHAT IS KNOWN ALREADY
The prevalence of PCOS in Iran is relatively high and studying QOL in these patients is important. The PCOSQ has previously been translated and validated in Iran but no studies had examined the psychometric properties of the MPCOSQ in Iran.
STUDY DESIGN, SIZE, AND DURATION
This was a cross-sectional study. After linguistic validation of the Iranian version of MPCOSQ, an expert panel evaluated the items by assessing the content validity index (CVI) and content validity ratio (CVR). Then a semi-structured interview was conducted to assess face validity. Consequently, exploratory factor analysis (EFA) and confirmatory factor analysis (CFA) were performed to indicate the scale constructs (n = 200). In addition, reliability analyses including internal consistency and test–retest analysis were carried out.
PARTICIPANTS/MATERIALS, SETTING, AND METHODS
This study was carried out on women with PCOS (n = 200) who attended two private gynecology clinics in Kashan, Iran. Patients were eligible if they met each of the following criteria: 15–40 years of age; married; not having non-classic adrenal hyperplasia, thyroid dysfunction and hyperprolactinemia; Iranian; not having problems in speaking or listening; having two of the following Rotterdam diagnostic criteria: (i) polycystic ovaries visualized on ultrasound scan (presence of 12 follicles or more in one or both ovaries and/or increased ovarian volume >10 ml), (ii) clinical signs of hyperandrogenism (the hirsutism score based on the Ferriman–Gallwey score >7 or obvious acne) and/or an elevated plasma testosterone (normal range testosterone <2 nmol/l), (iii) having an interval between menstrual periods >35 days and/or amenorrhea as the absence of vaginal bleeding for at least 6 months, i.e. 199 days.
MAIN RESULTS AND THE ROLE OF CHANCE
CVI and CVR scores for MPCOSQ were 0.96 and 0.92, respectively. A six-factor solution (emotional disturbance, weight, infertility, acne, menstrual difficulties and hirsutism) emerged as a result of an EFA and explained 0.64% of the variance observed. The result of the EFA supported the item ‘late menstrual period’ being placed in the menstrual rather than the emotional subscale. The results of the CFA for six-factor model for MPCOSQ indicated an acceptable fit for the proposed model. Additional analyses indicated satisfactory results for internal consistency (Cronbach's alpha ranging from 0.76 to 0.92) and intraclass correlation coefficients (ranging from 0.71 to 0.92). Moving ‘late menstrual period’ from the emotional to the menstrual subscale significantly improved the reliability coefficient for both subscales.
LIMITATIONS, REASONS FOR CAUTION
Study of patients with PCOS attending two private gynecology clinics may limit generalization of the findings to the entire population with PCOS. All patients in this study were married for cultural reasons in Iran. Therefore, the results of the present study have to be interpreted with some caution.
WIDER IMPLICATIONS OF THE FINDINGS
The Iranian version of the MPCOSQ will fill an important gap in measuring the QOL in patients with PCOS in the research and community settings in Iran.
STUDY FUNDING/COMPETING INTEREST(S)
The authors declare that they have no conflict of interest.
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Psychological stress and reproductive aging among pre-menopausal women
BACKGROUND
Life history models suggest that biological preparation for current versus longer term reproduction is favored in environments of adversity. In this context, we present a model of reproductive aging in which environmental adversity is proposed to increase the number of growing follicles at the cost of hastening the depletion of the ovarian reserve over time. We evaluated this model by examining psychological stress in relation to reproductive aging indexed by antral follicle count (AFC), a marker of total ovarian reserve. We hypothesized that stress would be related to (i) higher AFC in younger women, reflecting greater reproductive readiness as well as (ii) greater AFC loss across women, reflecting more accelerated reproductive aging.
METHODS
In a multi-ethnic, community sample of 979 participants [ages 25–45 (mean (standard deviation) = 35.2 (5.5)); 27.5% Caucasian] in the Ovarian Aging study, an investigation of the correlates of reproductive aging, the interaction of age-x-stress was assessed in relation to AFC to determine whether AFC and AFC loss varied across women experiencing differing levels of stress. Stress was assessed by the perceived stress scale and AFC was assessed by summing the total number of antral follicles visible by transvaginal ultrasound.
RESULTS
In linear regression examining AFC as the dependent variable, covariates (race/ethnicity, socio-economic status, menarcheal age, hormone-containing medication for birth control, parity, cigarette smoking, bodymass index, waist-to-hip ratio) and age were entered on step 1, stress on step 2 and the interaction term (age-x-stress) on step 3. On step 3, significant main effects showed that older age was related to lower AFC (b = –0.882, P = 0.000) and greater stress was related to higher AFC (b = 0.545, P = 0.005). Follow-up analyses showed that the main effect of stress on AFC was present in the younger women only. A significant interaction term (b = –0.036, P = 0.031) showed the relationship between age and AFC varied as function of stress. When the sample was divided into tertiles of stress, the average follicle loss was –0.781, –0.842 and –0.994 follicles/year in the low-, mid- and high-stress groups, respectively.
CONCLUSIONS
Psychological stress was related to higher AFC among younger women and greater AFC decline across women, suggesting that greater stress may enhance reproductive readiness in the short term at the cost of accelerating reproductive aging in the long term. Findings are preliminary, however, due to the cross-sectional nature of the current study.
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National survey of the current management of endometriomas in women undergoing assisted reproductive treatment
STUDY QUESTION
What is the current management of women with ovarian endometriomas undergoing assisted reproductive treatment (ART) in the UK?
SUMMARY ANSWER
It appears that the majority of gynaecologists in the UK offer surgery (mostly cystectomy) for endometriomas prior to ART, regardless of the presence of symptoms.
WHAT IS KNOWN ALREADY
The ideal management of endometriomas in women undergoing ART remains controversial and presents a dilemma to reproductive specialists.
STUDY DESIGN, SIZE AND DURATION
This was a national cross-sectional survey. A total of 388 gynaecologists completed the questionnaire.
PARTICIPANTS, SETTINGS AND METHODS
All clinicians fully registered with the Royal College of Obstetricians and Gynaecologists were contacted. An 11-item survey was administered electronically using Survey Monkey software. Quantitative data were analysed using descriptive and comparative statistics.
MAIN RESULTS AND THE ROLE OF CHANCE
The majority of responders were consultants (65%), 25% practiced ART and 65% performed laparoscopic surgery. Overall, 95% of responders would offer surgery for endometriomas in women undergoing ART, either on the basis of the size (>3–5 cm) of the endometrioma (52%), the presence of symptoms (16%), the presence of multiple/bilateral endometriomas (2%), regardless of the size and symptoms (19%) or only to women undergoing IVF (6%). The remaining 5% of responders would not offer surgery before ART. Excision was the most common surgical modality (68%), followed by ablation (25%). Laparoscopic surgeons were almost twice as likely to ‘offer surgery to all patients with endometriomas prior to ART’ compared with clinicians performing laparotomy (22 versus 12%, P < 0.001).
LIMITATIONS, REASONS FOR CAUTION
Our overall response rate, with answers to the questionnaire, was low (15%). However, the response rate amongst reproductive specialists was estimated at 60%. It is possible that there might have been an element of bias towards over-representation of responders who are more concerned about ‘normalization’ of the pelvic anatomy. Furthermore, our survey relied on self-reporting of practice and it is possible that being presented with a list of ‘ideal’ options may have resulted in respondent bias.
WIDER IMPLICATIONS OF THE FINDINGS
Despite the available evidence that surgery for endometriomas does not improve the outcome of ART and may damage ovarian reserve, it seems that the majority of gynaecologists in the UK offer ovarian cystectomy to their patients.
STUDY FUNDING/COMPETING INTERESTS
None.
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Evaluation of a panel of 28 biomarkers for the non-invasive diagnosis of endometriosis
Background
At present, the only way to conclusively diagnose endometriosis is laparoscopic inspection, preferably with histological confirmation. This contributes to the delay in the diagnosis of endometriosis which is 6–11 years. So far non-invasive diagnostic approaches such as ultrasound (US), MRI or blood tests do not have sufficient diagnostic power. Our aim was to develop and validate a non-invasive diagnostic test with a high sensitivity (80% or more) for symptomatic endometriosis patients, without US evidence of endometriosis, since this is the group most in need of a non-invasive test.
Methods
A total of 28 inflammatory and non-inflammatory plasma biomarkers were measured in 353 EDTA plasma samples collected at surgery from 121 controls without endometriosis at laparoscopy and from 232 women with endometriosis (minimal–mild n = 148; moderate–severe n = 84), including 175 women without preoperative US evidence of endometriosis. Surgery was done during menstrual (n = 83), follicular (n = 135) and luteal (n = 135) phases of the menstrual cycle. For analysis, the data were randomly divided into an independent training (n = 235) and a test (n = 118) data set. Statistical analysis was done using univariate and multivariate (logistic regression and least squares support vector machines (LS-SVM) approaches in training- and test data set separately to validate our findings.
Results
In the training set, two models of four biomarkers (Model 1: annexin V, VEGF, CA-125 and glycodelin; Model 2: annexin V, VEGF, CA-125 and sICAM-1) analysed in plasma, obtained during the menstrual phase, could predict US-negative endometriosis with a high sensitivity (81–90%) and an acceptable specificity (68–81%). The same two models predicted US-negative endometriosis in the independent validation test set with a high sensitivity (82%) and an acceptable specificity (63–75%).
Conclusions
In plasma samples obtained during menstruation, multivariate analysis of four biomarkers (annexin V, VEGF, CA-125 and sICAM-1/or glycodelin) enabled the diagnosis of endometriosis undetectable by US with a sensitivity of 81–90% and a specificity of 63–81% in independent training- and test data set. The next step is to apply these models for preoperative prediction of endometriosis in an independent set of patients with infertility and/or pain without US evidence of endometriosis, scheduled for laparoscopy.
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