BACKGROUND

There is an increasing body of evidence that human pluripotent stem cells (hPSCs) are prone to (epi)genetic instability during in vitro culture. This review aims at giving a comprehensive overview of the current knowledge on culture-induced (epi)genetic alterations in hPSCs and their phenotypic consequences.

METHODS

Combinations of the following key words were applied as search criteria: human induced pluripotent stem cells and human embryonic stem cells in combination with malignancy, tumorigenicity, X inactivation, mitochondrial mutations, genomic integrity, chromosomal abnormalities, culture adaptation, aneuploidy and CD30. Only studies in English, on hPSCs and focused on (epi)genomic integrity were included. Further manuscripts were added from cross-references.

RESULTS

Numerous (epi)genetic aberrations have been detected in hPSCs. Recurrent genetic alterations give a selective advantage in culture to the altered cells leading to overgrowth of abnormal, culture-adapted cells. The functional effects of these alterations are not yet fully understood, but suggest a (pre)malignant transformation of abnormal cells with decreased differentiation and increased proliferative capacity.

CONCLUSIONS

Given the high degree of (epi)genetic alterations reported in the literature and altered phenotypic characteristics of the abnormal cells, controlling for the (epi)genetic integrity of hPSCs before any clinical application is an absolute necessity.

Source:
http://humupd.oxfordjournals.org/cgi/content/short/19/2/187?rss=1

Mitochondrial medicine is one of the few areas of genetic disease where germ-line transfer is being actively pursued as a treatment option. All of the germ-line transfer methods currently under development involve some carry-over of the maternal mitochondrial DNA (mtDNA) heteroplasmy, potentially delivering the pathogenic mutation to the offspring. Rapid changes in mtDNA heteroplasmy have been observed within a single generation, and so any ‘leakage’ of mutant mtDNA could lead to mtDNA disease in future generations, compromising the reproductive health of the first generation, and leading to repeated interventions in subsequent generations. To determine whether this is a real concern, we developed a model of mtDNA heteroplasmy inheritance by studying 87 mother–child pairs, and predicted the likely outcome of different levels of ‘mutant mtDNA leakage’ on subsequent maternal generations. This showed that, for a clinical threshold of 60%, reducing the proportion of mutant mtDNA to <5% dramatically reduces the chance of disease recurrence in subsequent generations, but transmitting >5% mutant mtDNA was associated with a significant chance of disease recurrence. Mutations with a lower clinical threshold were associated with a higher risk of recurrence. Our findings provide reassurance that, at least from an mtDNA perspective, methods currently under development have the potential to effectively eradicate pathogenic mtDNA mutations from subsequent generations.

Source:
http://humrep.oxfordjournals.org/cgi/content/short/28/3/554?rss=1

The field of gamete donation for medically assisted reproduction purposes is evolving. While anonymous gamete donation was long the preferred practice, a new focus on the rights and interests of donor-conceived children has led a number of countries to shift towards an open-identity system. However, this evolution appears to overlook whether information exchange could also be of interest to the other parties involved, in particular the gamete donors. In this article, we analyse the question whether donors should be granted a right to some information about the offspring conceived by their donations. We constructed five arguments which donors could use in support of such a claim: (i) It can be of great importance to the donors' and their own children's health that they receive medical information (in particular, evidence of an unsuspected genetic disease) about the donor offspring; (ii) basic information (such as whether any children were born) could be a way to acknowledge donors for their altruistic behaviour; (iii) general information (information about the child's wellbeing) about the donor offspring could ease the donors' potential concern about and sense of responsibility for the offspring; (iv) basic information could provide an important enrichment of the donors' identities; (v) identifying information would be useful for donors who want to contact the donor offspring. No strong arguments in favour of granting donors the right to identifying information were found. An exchange of this type of information should only be accepted when all parties agree. Taken together, the four first arguments form a strong case for granting donors a right to several types of anonymous information about the donor offspring.

Source:
http://humrep.oxfordjournals.org/cgi/content/short/28/3/560?rss=1

STUDY QUESTION

Does vitrification of human immature testicular tissue (ITT) have potential benefits for future fertility preservation? Does vitrification of human ITT have potential benefits in an in vivo murine xenotransplantation model?

SUMMARY ANSWER

Vitrification is able to maintain proliferation capacity in spermatogonial cells after 6 months of xenografting.

WHAT IS KNOWN ALREADY

Controlled slow-freezing is the procedure currently applied for ITT cryobanking in clinical practice. Vitrification has been proposed as a promising technique for long-term storage of ITT, with a view to preserving spermatogonial stem cells (SSCs) for future fertility restoration in young boys suffering from cancer. After vitrification of ITT, in vitro survival of SSCs was demonstrated, but their functionality was not evaluated.

STUDY DESIGN, SIZE, DURATION

Ten ITT pieces issuing from 10 patients aged 2–12 years were used. Fragments of fresh tissue (serving as controls) and fresh, frozen-thawed and vitrified-warmed testicular pieces xenografted to the scrotum of nude mice for 6 months were compared.

MATERIALS, SETTING, METHODS

Upon graft removal, histological and immunohistochemical analyses were performed to evaluate spermatogonia (SG) (MAGE-A4), intratubular proliferation (Ki67), proliferating SG and Leydig cells (3β-HSD). The entire piece of grafted tissue was assessed in each case.

MAIN RESULTS AND THE ROLE OF CHANCE

Seminiferous tubules showed good integrity after cryopreservation and xenografting for 6 months in all three groups. Survival of SG and their ability to proliferate was observed by immunohistochemistry in all grafted groups. SG were able to initiate spermatogenesis, but blockage at the pachytene stage was observed. The recovery rate of SG was 3.4 ± 3.8, 4.1 ± 7.3 and 7.3 ± 6.3%, respectively, for fresh, slow-frozen and vitrified-warmed tissue after 6 months of xenografting.

LIMITATIONS, REASONS FOR CAUTION

The study is limited by the low availability of ITT samples of human origin. The mouse xenotransplantation model needs to be refined to study human spermatogenesis.

WIDER IMPLICATIONS OF THE FINDINGS

The findings of the present study have potential implications for cryobanking of ITT and fertility preservation. Spermatogonial loss recorded after fresh ITT transplantation indicates that the avascular grafting technique needs to be optimized. There are so far no convincing data justifying modification of current clinical practice for ITT storage with slow-freezing, but this study demonstrates that it is worth pursuing optimization of ITT vitrification as an alternative for preservation of SSCs.

STUDY FUNDING/COMPETING INTEREST(S)

The present study was supported by a grant from the Fonds National de la Recherche Scientifique de Belgique (grant Télévie N^° 7. 4.572.09.F). The authors declare that there is no conflict of interest.

Source:
http://humrep.oxfordjournals.org/cgi/content/short/28/3/578?rss=1

STUDY QUESTION

What are the reference values for delineating altered somatic cell gene expression from transcript enrichment/dilution in gene expression studies of human spermatogenesis?

SUMMARY ANSWER

We have designed a crosstable and rule-of-thumb values for different stages of spermatogenic impairment that define the reference cut-off values for altered gene expression in Sertoli and Leydig cells in the context of impaired spermatogenesis.

WHAT IS KNOWN ALREADY

Morphometrical studies have shown that on the cellular level, impaired spermatogenesis results in a relative enrichment of somatic cell types. However, until now it is not known how this affects transcript levels in gene expression studies.

STUDY DESIGN, SIZE DURATION

In this study, 31 testis biopsies from men with different stages of spermatogenic impairment (full spermatogenesis, hypospermatogenesis, meiotic arrest, spermatogonial presence, Sertoli cell-only syndrome, complete tubular atrophy) were used to define reference ratios of somatic transcript enrichment/dilution. The reference ratios were validated on an independent test set of 28 samples and on gene expression data from men with Y-chromosomal microdeletions.

PARTICIPANTS/MATERIAL, SETTING, METHODS

High-quality microarray data were filtered with respect to Sertoli- and Leydig-cell-specific genes. General reference enrichment/dilution factors for these two cell types for all combinations of spermatogenic impairment were calculated using robust permutation statistics. To validate the specificity of the filtered transcripts, we calculated ratios for an independent test set of spermatogenic impairment and for transcriptional data from men with Y-chromosomal microdeletions, and checked the functional enrichment (gene ontology) and cellular localization of the corresponding proteins in a histological database and assessed their correlation with testicular size.

MAIN RESULTS AND THE ROLE OF CHANCE

Filtering of Sertoli- and Leydig-cell-specific genes resulted in a set of 54 and 332 transcripts, respectively. These were used in defining robust reference dilution/enrichment factors of somatic transcripts for all spermatogenic levels and were compiled in a reference crosstable. Validation on an independent test set showed ratios within 0.5 units of our reference crosstable. Analysis of the resulting transcripts with respect to functional enrichment for Sertoli- and Leydig-cell-specific functions and protein expression, as obtained from an immunohistochemical database, indicated filtering of data sets highly enriched for Sertoli and Leydig cell function. The dilution/enrichment ratios differed significantly when transcripts were interrogated in samples with Y-chromosomal microdeletions, pointing to an overall decreased expression of somatic markers in a genetically altered background.

LIMITATIONS, REASONS FOR CAUTION

The defined reference ratios might apply with some restrictions in samples that display very heterogeneous histology (e.g. Sertoli cell only with a significant proportion of spermatogenic foci) or when spermatogenic impairment is a consequence of an altered genetic background.

WIDER IMPLICATIONS OF THE FINDINGS

The reference dilution/enrichment values for somatic testicular transcripts as defined in this study are to be seen as cut-off values for discriminating between simple transcript dilution/enrichment as a consequence of an altered germ cell composition and actual transcriptional regulation. Future studies dealing with transcriptional changes in testicular somatic cells in a background of altered germ cell quantities should consider these correction factors in order to avoid the description of transcriptional changes that are based simply on shifts in somatic cellular quantities.

STUDY FUNDING/COMPETING INTEREST(S)

Financial support was from grant Sp721/1-3 of the German Research Foundation. There are no competing interests to be declared.

Source:
http://humrep.oxfordjournals.org/cgi/content/short/28/3/590?rss=1

STUDY QUESTION

What is the role of carbohydrates in the binding of human sperm to the zona pellucida (ZP) and what are potential implications for pathogenesis?

SUMMARY ANSWER

Both lectin-like and protein–protein interactions play an essential role in human gamete interactions.

WHAT IS KNOWN ALREADY

Studies in the mouse and human indicate a role for both lectin-like and protein–protein interactions during sperm binding to the ZP.

STUDY DESIGN, SIZE, DURATION

Non-systematic literature review.

MAIN RESULTS AND THE ROLE OF CHANCE

Ultrasensitive analysis by mass spectrometry of glycans linked to the human ZP has confirmed that this matrix is coated with a high density of complex type N-glycans terminated with the sialyl-Lewis^x (sLe^x) sequence, the universal selectin ligand. Selectins are essential for lymphocyte homing, and they participate in the initial binding of circulating leukocytes to activated endothelium at the sites of infection and tissue injury. Subsequent inhibition studies confirmed that either the sLe^x tetrasaccharide or neoglycoproteins terminated with this sequence inhibited human sperm–ZP binding by 70% in the hemizona assay. These results support the hypothesis that both lectin-like and protein–protein interactions play an essential role in human gamete interactions. The sLe^x sequence is also a ligand for siglec-9, a lectin-bearing immunoreceptor tyrosine-based inhibitory motif that transmits inhibitory signals. This siglec is expressed on a wide variety of different types of human leukocytes and lymphocytes. This result is consistent with the hypothesis that human ZP glycans are also being employed for immune recognition of the egg and the histoincompatible embryo prior to blastocyst hatching.

LIMITATIONS, REASONS FOR CAUTION

This field of study is complex and more experimental work is needed to reveal fully the mechanism of sperm–ZP binding and how it varies between species.

WIDER IMPLICATIONS OF THE FINDINGS

Knowledge about the glycans involved in sperm–egg binding may be relevant to infertility due to fertilization failure and also to the mother's immune tolerance of the preimplantation embryo.

STUDY FUNDING/COMPETING INTEREST(S)

Studies focused on human sperm–egg interactions carried out by the author and coworkers have been supported by the Life Sciences Mission Enhancement Reproductive Biology Program funded by the State of Missouri, a Research Board Grant (CB000500) supported by the University of Missouri System and a grant from the Jeffress Memorial Trust of Virginia. Support from the Breeden-Adams Foundation has also been obtained to investigate potential linkages to tumor evasion. The author has no conflict of interest to declare.

Source:
http://humrep.oxfordjournals.org/cgi/content/short/28/3/566?rss=1

STUDY QUESTION

Can we accurately define a group of pregnancies of unknown location (PULs) as low risk in order to safely reduce follow-up for these pregnancies and allocate resources to pregnancies at an increased risk of being ectopic?

SUMMARY ANSWER

Prediction model M4 classified around 70% of PULs as low risk, of which around 97% were later characterized as failed PULs or intrauterine pregnancies (IUPs), while still classifying 88% of ectopic pregnancies as high risk.

WHAT IS KNOWN ALREADY

Depending on the level of suspicion of ectopic pregnancy (EP), women with a PUL receive a lengthy follow-up in order to confirm the location and viability of the pregnancy.

STUDY DESIGN, SIZE, DURATION

A multi-centre diagnostic accuracy study of 1962 patients was carried out between 2003 and 2007 for retrospective temporal validation and between 2009 and 2011 for prospective external validation. The reference standard is the final characterization of PUL as failed pregnancies or IUPs (low risk), or as ectopic pregnancies (high risk). M4 is a multinomial logistic regression model based on the serum human chorionic gonadotrophin (hCG) levels at presentation and 48 h later.

PARTICIPANTS/MATERIALS, SETTING, METHODS

Temporal validation data from 1341 PULs collected at St George's Hospital in London were available, of which 53% were failed, 39% were intrauterine and 8% were ectopic pregnancies. External validation data from 621 PULs collected at four other London-based teaching hospitals were available, of which 63% were failed, 22% were intrauterine and 15% were ectopic pregnancies.

MAIN RESULTS AND THE ROLE OF CHANCE

The EP rate varied between 8 and 16% across the five hospitals. At St George's, 980 [73.1%, 95% confidence interval (CI): 70.5–75.4] PULs were considered low risk. Of these, 963 were failed PULs or IUPs (98.3%, 95% CI: 97.2–98.9) and 17 were ectopic pregnancies. At the other four hospitals, 62–75% were considered low risk, with 96–98% of these turning out to be failed PUL or IUP. Eighty-five percent (95% CI: 76.8–90.2) of the ectopic pregnancies were considered high risk at St George's, compared with 80–92% in the other hospitals.

LIMITATIONS, REASONS FOR CAUTION

Of total, 120 patients had been excluded due to loss to follow-up, and a further 102 patients because of missing hCG levels due to differences in local clinical practice. There are variations in the definition of a PUL used in different countries.

WIDER IMPLICATIONS OF THE FINDINGS

The suggested protocol could safely reduce the follow-up in the majority of PUL such that units could increase the focus on women at a risk of complications. This would lead to a change in the management of the majority of women with a PUL and a more efficient use of resources. At the end of the manuscript, we provide a link to enable clinicians to use the protocol.

STUDY FUNDING/COMPETING INTEREST(S)

B.V.C. is supported by a postdoctoral fellowship from the Research Foundation Flanders (FWO). K.V.H. is supported by a fellowship from the Flanders' Agency for Innovation by Science and Technology (IWT-Vlaanderen), by the Research Council KU Leuven (GOA MaNet), by the Flemish Government (iMinds) and by the Belgian Federal Science Policy Office (IUAP P7/DYSCO). T.B. is supported by the Imperial Healthcare NHS Trust NIHR Biomedical Research Centre. No competing interests are declared.

Source:
http://humrep.oxfordjournals.org/cgi/content/short/28/3/609?rss=1

STUDY QUESTION

Is exposure to perfluorinated compounds (PFCs) associated with testicular function (reproductive hormone levels and semen quality) in healthy men?

SUMMARY ANSWER

PFOS levels were significantly negatively associated with serum testosterone (total and calculated free), but not with any other reproductive hormones or semen quality.

WHAT IS KNOWN ALREADY

In animals, some PFCs have endocrine disrupting potential, but few studies have investigated PFCs in relation to human testicular function. Previously, we and others have observed a negative association between serum PFC levels and sperm morphology. The potential associations with reproductive hormones remain largely unresolved.

STUDY DESIGN, SIZE, DURATION

A cross-sectional study of 247 men was conducted during 2008–2009.

PARTICIPANTS/MATERIALS, SETTING, METHODS

Healthy men from the general population, median age of 19 years, gave serum and semen samples. Serum samples were analysed for total testosterone (T), estradiol (E), sex hormone-binding globulin (SHBG), luteinizing hormone (LH), follicle-stimulating hormone (FSH) and inhibin-B and 14 PFCs, including perfluorooctanesulfonate (PFOS). Semen samples were analysed according to the WHO criteria.

MAIN RESULTS AND THE ROLE OF CHANCE

PFOS levels were negatively associated with testosterone (T), calculated free testosterone (FT), free androgen index (FAI) and ratios of T/LH, FAI/LH and FT/LH. Other PFCs were found at lower levels than PFOS and did not exhibit the same associations. PFC levels were not significantly associated with semen quality. PFOS levels in these samples collected in 2008–2009 were lower than in our previous study of men participating in 2003.

LIMITATIONS, REASONS FOR CAUTION

Results were robust to adjustment for relevant confounders; however, the possibility of chance associations due to multiple testing or effects of uncontrolled confounding cannot be ruled out.

WIDER IMPLICATIONS OF THE FINDINGS

Our previous findings of decreased sperm morphology in the most highly PFC exposed men were not replicated, possibly due to a lack of highly exposed individuals; however, a recent independent study also did corroborate such an inverse association. The negative association between serum PFOS and testosterone indicates that testosterone production may be compromised in individuals with high PFOS exposure.

STUDY FUNDING/COMPETING INTEREST(S)

The authors received financial support from the European Commission (DEER, FP7-2007-212844), the Danish Agency for Science, Technology and Innovation (grant nos. 27107068 and 09-067180), Rigshospitalet (grant no. 961506336), the University of Copenhagen, the Danish Ministry of Health and the Danish Environmental Protection Agency (MST-621-00013), and Kirsten and Freddy Johansen Foundation (grant no. 95-103-72087). The funding organizations played no role in the design and conduct of the study, in collection, management, analysis and interpretation of the data; or in the presentation, review or approval of the manuscript. The authors declare that they have no competing financial interests.

Source:
http://humrep.oxfordjournals.org/cgi/content/short/28/3/599?rss=1

STUDY QUESTION

Is there a link between morphometric characteristics measured by a computer-assisted scoring system and clinical pregnancy outcome?

SUMMARY ANSWER

The results confirm that computer-assisted assessment of the total embryo volume is associated with clinical pregnancy outcome and can be used to complement current procedures of embryo selection.

WHAT IS KNOWN ALREADY

Morphometric analysis of a large group of embryos has revealed the potential to optimize algorithms for image-analysis systems for the grading of embryos and predicting pregnancy outcomes.

STUDY DESIGN, SIZE, DURATION

Oocytes and embryos were obtained from 458 patients who underwent single embryo transfer on Day 3 after IVF/ICSI, between September 2006 and December 2010 at the Leuven University Fertility Center, Belgium. In total, the data set contained 2796 embryos including 458 embryos that were transferred on Day 3. Ongoing pregnancy was defined as the presence of at least one intrauterine gestational sac at 20 weeks.

PARTICIPANTS/MATERIALS, SETTING, METHODS

Patients included in this study were younger than 36 years, entering their first (n = 375) or second (n = 83) IVF/ICSI cycle and were only included once. Patients were excluded if the cycle included biopsy for PGD or if donor sperm/donor oocytes were used. Based on the 26 sequential images of the same embryo taken at one time point in different planes, the software calculates the total cytoplasmic volume for each time point, from which any reduction or change in the volume with time can be assessed (which helps interpret the degree of fragmentation) and the size of blastomeres. The diameter of the smallest and largest blastomere and the total volume of each embryo were extracted from the computer-assisted scoring system database and the coefficient of diversity was calculated for Days 1, 2 and 3. A logistic regression analysis was performed to determine the range of embryo volume associated with an increased chance of pregnancy.

MAIN RESULTS AND THE ROLE OF CHANCE

On Day 3, blastomeres of 8-cell stage embryos were less divergent in size than those of 6-, 7-, 9-cell stage embryos. Although, the coefficients of diversity (ratio of the largest:smallest blastomeres) of implanted embryos tended to be lower than for non-implanted embryos, the difference was only significant for 6-cell stage embryos (P = 0.02). After logistic regression, an association between total embryo volume and pregnancy was observed which had a quadratic nature: both lower and higher volumes were associated with a lower probability of successful pregnancy. A significant association was identified between total embryo volume and pregnancy rate on both Days 2 (P = 0.003) and 3 (P = 0.0003). Diagnostic measures (sensitivity, specificity, positive predictive value, accuracy and c-statistics) of the defined volume range were relatively poor. However, results showed a good negative predictive value [76.86% (95% confidence interval 71.03–82.02) on Day 3].

LIMITATIONS, REASONS FOR CAUTION

A general disadvantage of studies evaluating the impact of a characteristic on the implantation potential of an embryo is the fact that the best embryo is chosen for transfer. No comparisons can therefore be made with the other embryos. Moreover, the decision process is currently based on a non-automated, standard scoring system, which means that a ‘bias’ in the selection process is always present.

WIDER IMPLICATIONS OF THE FINDINGS

Our results are an important step towards the development of an automated computer-assisted scoring system for the morphological characteristics of human embryos to improve embryo selection for optimizing implantation potential. Total embryo volume appears to be one of the objective characteristics that should be included.

STUDY FUNDING/COMPETING INTEREST(S)

None.

TRIAL REGISTRATION NUMBER

Not applicable.

Source:
http://humrep.oxfordjournals.org/cgi/content/short/28/3/627?rss=1

STUDY QUESTION

Are the morphokinetics of growing embryos affected by the type of culture media utilized?

SUMMARY ANSWER

Morphokinetic parameters used for embryo selection are not affected between the two different concept culture media analyzed.

WHAT IS KNOWN ALREADY

Studies on the effect of culture media on human embryos have focused on evaluating different in-house and commercially available media as well as comparing outcomes among different commercial media. Nonetheless, the evaluation of embryo development in these studies was based on static observations and very little is known from a dynamic point of view.

STUDY DESIGN, SIZE, DURATION

Prospective cohort study, October 2010 and April 2011.

PARTICIPANTS/MATERIALS, SETTING, METHODS

University-affiliated infertility center. Patients undergoing egg donation (n = 75) in which embryos were cultured with two different types of media in a time-lapse system. Embryo development was analyzed with time-lapse imaging for single step media (Global^®) and sequential media (Sage^® Cleavage). Variables studied included the timing to two cells (t2), three cells (t3), four cells (t4) and five cells (t5) as well as the length of the second cell cycle (cc2 = t3 – t2) and the synchrony in the division from two to four cells (s2 = t4 – t3). Implantation and clinical pregnancy rates were also analyzed.

MAIN RESULTS AND THE ROLE OF CHANCE

No statistically significant differences were observed between the two media for all the variables analyzed. When analyzing the percentage of embryos falling within the optimal ranges proposed for s2, cc2 and t5, we did not find significant differences between the two media. Pregnancy and implantation rates were similar for the three types of transfers: 48.0% (CI 95% 28.4–67.6) and 42.0% (CI 95% 22.5–61.4) with Global media; 58.8% (CI 95% 35.4–82.2) and 38.2% (CI 95% 15.0–61.4) with Cleavage media; and 58.1% (CI 95% 40.7–75.4) and 37.1% (CI 95% 22.1–52.1) with mixed transferred, respectively. Multiple implantations (twins) were also similar among the three groups, with 24.0% (CI 95% 9.3–45.1) for transfers with embryos cultured in Global media, 17.6% (CI 95% 3.7–43.3) for transfers with embryos cultured in Cleavage media and 22.5% (CI 95% 9.5–41.0) with mixed transfers.

LIMITATIONS, REASONS FOR CAUTION

The study was not powered to test differences in pregnancy rates between the two culture media, as this was not the hypothesis tested. Results are based on observations with embryos from oocyte donors and need to be repeated with embryos from infertile patients of different ages.

WIDER IMPLICATIONS OF THE FINDINGS

The absence of differences in morphokinetics between two different media concepts validates the algorithm for embryo selection in diverse culture conditions.

STUDY FUNDING/COMPETING INTEREST(S)

No specific funding was obtained for this study; it was solely funded by IVI. None of the authors have any economic affiliation with Unisense Fertilitech A/S but IVI is a minor shareholder in Unisense Fertilitech A/S.

TRIAL REGISTRATION NUMBER

Not applicable.

Source:
http://humrep.oxfordjournals.org/cgi/content/short/28/3/634?rss=1

STUDY QUESTION

Does high-magnification sperm selection influence oocyte fertilization and further embryo development?

SUMMARY ANSWER

The present study did not show a difference in oocyte fertilization rate, nor in embryo development between high-magnification intracytoplasmic morphologically selected sperm injection (IMSI) and conventional ICSI.

WHAT IS KNOWN ALREADY

The presence of nuclear vacuoles in sperm seems to influence embryo development and more specifically blastocyst formation. The use of high magnification for morphological sperm selection prior to ICSI has been associated with higher pregnancy rates and lower miscarriage rates.

STUDY DESIGN, SIZE, DURATION

A prospective sibling-oocyte study was conducted, including 350 ICSI cycles to alleviate male infertility. Cycles were included from March 2010 to November 2011.

PARTICIPANTS/MATERIALS, SETTING, METHODS

On the day of treatment, a high-magnification sperm morphology was assessed on at least 200 spermatozoa. Primary endpoints were oocyte fertilization rate and embryo development. Because embryo transfers were not randomized, the clinical outcome (clinical pregnancy rate per transfer cycle) was descriptive. However, the embryologist selecting the embryos for transfer was blinded for the sperm selection procedure.

MAIN RESULTS AND THE ROLE OF CHANCE

IMSI morphology was assessed in 330 semen samples, resulting in the following distribution: 18.1 ± 14.8% Grade I, 15.2 ± 10.3% Grade II, 12.3 ± 9.1% Grade III and 54.4 ± 23.2% Grade IV. Oocyte fertilization rate was 79.1 and 77.3% after IMSI and ICSI, respectively (NS, paired t-test). Embryo development was similar in both treatment groups up to Day 5 of preimplantation development. Comparable numbers of IMSI-only (n = 125) and ICSI-only (n = 139) embryo transfers were performed. Clinical pregnancies with fetal heart beat were equally distributed over transfers with embryos from IMSI-only (34.4%) or ICSI-only treatment (36.7%).

LIMITATIONS, REASONS FOR CAUTION

The clinical outcome remains descriptive. No firm conclusions could be drawn on cycle rank as a possible indication for IMSI.

WIDER IMPLICATIONS OF THE FINDINGS

The prevalence of vacuoles in normal-shaped spermatozoa is as low as 27.5%. A routine application of IMSI in unselected artificial reproductive technology patients cannot be advocated.

STUDY FUNDING/COMPETING INTEREST(S)

None.

Source:
http://humrep.oxfordjournals.org/cgi/content/short/28/3/617?rss=1

STUDY QUESTION

What is the predictive value of pregnancy intentions on contraceptive behaviours among women aged 18–19?

SUMMARY ANSWER

Women aged 18–19 have high levels of inconsistent use of contraception, which mostly occur at times when women strongly wish to avoid a pregnancy.

WHAT IS KNOWN ALREADY

Pregnancy intentions provide an indication of how well individuals achieve their reproductive goals. However, retrospective accounts of pregnancy intentions using dichotomous indicators suffer temporal instability and fail to capture the wide range of attitudes towards pregnancy.

STUDY DESIGN, SIZE, DURATION

In this study, data are drawn from a population-based survey of 992 women of ages 18–19 years in Michigan, who completed weekly journals assessing contraceptive use, pregnancy intentions and reproductive outcomes during 2.5 years of follow-up. The response rate was 86% for the baseline interview and 65% after 2.5 years of follow-up.

PARTICIPANTS/MATERIALS, SETTING, METHODS

We examined 15 446 pairs of journal entries. We used logistic regression with random effects to assess the predictive effect of women's desire to become pregnant and to avoid a pregnancy, measured each week, on consistency of use of contraception the following week.

MAIN RESULTS AND THE ROLE OF CHANCE

Women reported inconsistent use of contraception in more than a quarter of weekly journals (28.3%). Consistent use of contraception increased from 22 to 78% as women s intentions to become pregnant decreased and increased from 23 to 78% as motivations to avoid pregnancy increased. The combination of scores of the pregnancy desire and avoidance scales shows indifferent or ambivalent pregnancy attitudes in 8.6% of weekly records. These women were more likely to report inconsistent contraceptive use compared with women who expressed anti-conception attitudes [OR = 2.8 (2.2–3.5)]. However, 23% of women who had unequivocal anti-conception feelings did not use contraception consistently, contributing to 72% of the weeks of inconsistent use in our population.

LIMITATIONS, REASONS FOR CAUTION

In this study, consistency of contraceptive use, based on the use of contraception at every act of intercourse, does not fully capture a women's risk of becoming pregnant. The 35% attrition after 2.5 years may have affected the internal validity of our results, although a reanalysis based on the first year of observation produced very similar results.

WIDER IMPLICATIONS OF THE FINDINGS

Because most instances of inconsistent use of contraception occur among women who are keen to avoid a pregnancy, our results suggest there is room for improving contraceptive behaviours by promoting use of methods which do not require user adherence.

STUDY FUNDING/COMPETING INTEREST(S)

This work was supported by the National Institute of Child Health and Human Development for grant #R01-HDHD050329 (P.I. Barber, University of Michigan) and grant #R24HD047879 (Center infrastructure of the Office of Population Research at Princeton University, JT and KSH). None of the authors have a competing interest.

Source:
http://humrep.oxfordjournals.org/cgi/content/short/28/3/642?rss=1

STUDY QUESTION

Are levels of circulating angiogenic cells (CACs) affected by the presence of endometriosis?

SUMMARY ANSWER

Levels of CACs are equivalent in women with and without endometriosis.

WHAT IS KNOWN ALREADY

Murine models have suggested a role for CACs in the development of endometriosis, but their levels in humans have not yet been studied.

STUDY DESIGN, SIZE, DURATION

Eighty-seven women participated in this study. Recruitment took place from July 2010 to May 2012.

PARTICIPANTS/MATERIALS, SETTING, METHODS

All women underwent laparoscopy for investigation of symptoms suggestive of endometriosis. Thirty women had no evidence of endometriosis, and 47 women were found to have endometriosis at laparoscopy. CAC levels were determined in peripheral blood by flow cytometry in 64 women. Colony forming unit (CFU) analysis was conducted in 30 women. A separate group of 10 healthy, asymptomatic women donated blood at four time points to assess the effect of the menstrual cycle on CAC levels.

MAIN RESULTS AND THE ROLE OF CHANCE

For the whole sample, CAC levels (0.0797 ± 0.0052%) and CFU number (10.68 ± 1.98) were equivalent in women with and without endometriosis. CAC levels and CFU number were also unaffected by the stage of disease. No changes in CACs were detected during the menstrual cycle.

LIMITATIONS, REASONS FOR CAUTION

A difference of at least one standard deviation between the groups would be required to detect a difference with this sample size. Therefore, while CAC levels are not a useful biomarker of disease it is still possible that they are modestly altered by the presence of endometriosis. We did not describe specific types of lesion and it is possible that CAC elevation only occurs when vessel development is at its most prolific. Furthermore, although signals from endometriotic lesions may recruit CACs from blood, this may be insufficient to alter peripheral levels.

WIDER IMPLICATIONS OF THE FINDINGS

These data show that CACs are not a useful biomarker of endometriosis and indicate that they may be unaffected by the presence of this disease.

STUDY FUNDING/COMPETING INTEREST(S)

This work was supported by grants from the MRC (New Investigator Award, G0601458 to C.M.B.), the Oxford Partnership Comprehensive Biomedical Research Centre with funding from the Department of Health's NIHR Biomedical Research Centres Scheme and the Oxfordshire Health Services Research Committee (OHSRC). There are no conflicts of interest to be declared.

TRIAL REGISTRATION NUMBER

N/A.

Source:
http://humrep.oxfordjournals.org/cgi/content/short/28/3/651?rss=1

STUDY QUESTION

Can mixture survival models help distinguish infertility from subfertility in couples with an unexplained unfulfilled child wish?

SUMMARY ANSWER

Mixture models estimated that 47% of the couples were infertile; female age and previous pregnancy were significantly related to infertility, whereas duration of child wish was associated with a longer time to pregnancy for subfertile couples.

WHAT IS KNOWN ALREADY

To differentiate between couples who require assisted conception and couples who still have good chances of natural, i.e. unassisted, conception, several prediction models of natural conception have been developed. Prognostic factors in these models are usually assessed by Cox proportional hazard models that cannot differentiate between couples with an unfulfilled child wish who are subfertile, i.e. have reduced ability to conceive naturally, and couples who are really infertile, i.e. are completely unable to conceive naturally. We evaluated whether a mixture survival model can make such a distinction.

STUDY DESIGN, SIZE, DURATION

Consecutive couples presenting at the fertility clinics of 38 centres in the Netherlands between January 2002 and February 2004 joined a prospective cohort study. Of the 7860 couples in the cohort, 3917 couples met our inclusion criteria. The median follow-up was 219 days, with a maximum of 5 years.

PARTICIPANTS, SETTING, METHODS

Couples had to present with an unexplained cause of an unfulfilled child wish. A mixture model was used to estimate the proportion of couples who were infertile and the time to pregnancy for the subfertile couples.

MAIN RESULTS AND THE ROLE OF CHANCE

During the follow-up, 794 couples conceived naturally. The mixture model estimated that 47% [95% confidence interval (CI): 33–56%] of couples were infertile, despite the absence of objective factors indicating a cause for infertility. Of the evaluated prognostic factors, female age, duration of child wish, previous pregnancy, semen quality, BMI and cycle length, female age [odds ratio (OR): 1.11, 95% CI: 1.03–1.19] and previous pregnancy (0.22, 95% CI: 0.07–0.67) were significant predictors of infertility. Among subfertile couples, a longer duration of a child wish (FFR: 0.72, 95% CI: 0.61–0.85) was a significant prognostic factor for time to pregnancy. In the Cox models, all variables except BMI were significant predictors of time to pregnancy.

LIMITATIONS, REASONS FOR CAUTION

The mixture model had limited power due to a low number of couples at the end of the follow-up period. Mixture model analyses on external, long-term follow-up data are necessary to validate our results.

WIDER IMPLICATIONS OF THE FINDINGS

Mixture models could be a useful tool in selecting couples who require assisted reproductive technology because the effects of prognostic factors can be subdivided into effects on the fraction of infertile couples and effects on the time to pregnancy for subfertile couples, which is not possible in conventional models.

STUDY FUNDING/COMPETING INTEREST(S)

This study was supported by grant 945/12/002 from ZonMw, the Netherlands Organization for Health Research and Development, The Hague, the Netherlands.

Source:
http://humrep.oxfordjournals.org/cgi/content/short/28/3/658?rss=1

STUDY QUESTION

How do the different forms of regulation and public financing of IVF affect utilization in otherwise similar European welfare state systems?

SUMMARY ANSWER

Countries with more liberal social eligibility regulations had higher levels of IVF utilization, which diminished as the countries' policies became more restrictive.

WHAT IS KNOWN ALREADY

Europe is a world leader in the development and utilization of IVF, yet surveillance reveals significant differences in uptake among countries which have adopted different approaches to the regulation and and public financing of IVF.

STUDY DESIGN, SIZE, DURATION

A descriptive and comparative analysis of legal restrictions on access to IVF in 13 of the EU15 countries that affirmatively regulate and publicly finance IVF.

PARTICIPANTS/MATERIALS, SETTING, METHODS

Using 2009 data from the European Society of Human Reproduction and Embryology study of regulatory frameworks in Europe and additional legislative research, we examined and described restrictions on access to IVF in terms of general eligibility, public financing and the scope of available services. Multiple correspondence analysis was used to identify patterns of regulation and groups of countries with similar regulatory patterns and to explore the effects on utilization of IVF, using data from the most recent European and international IVF monitoring reports.

MAIN RESULTS AND THE ROLE OF CHANCE

Regulations based on social characteristics of treatment seekers who are not applicable to other medical treatments, including relationship status and sexual orientation, appear to have the greatest impact on utilization. Countries with the most generous public financing schemes tend to restrict access to covered IVF to a greater degree. However, no link could be established between IVF utilization and the manner in which coverage was regulated or the level of public financing.

LIMITATIONS, REASONS FOR CAUTION

Owing to the lack of data regarding the actual level of public versus private financing of IVF it is impossible to draw conclusions regarding equity of access. Moreover, the regulatory and utilization data were not completely temporally matched in what can be a quickly changing regulatory landscape.

WIDER IMPLICATIONS OF THE FINDINGS

Whether motivated by cost, eligility restrictions or the availability of particular services, cross-border treatment seeking is driven by regulatory policies, underscoring the extra-territorial implications of in-country political decisions regarding access to IVF.

STUDY FUNDING/COMPETING INTEREST(S)

There was no funding source for this study. The authors have no conflicts of interest to declare.

Source:
http://humrep.oxfordjournals.org/cgi/content/short/28/3/666?rss=1

STUDY QUESTION

Has the change in donor anonymity legislation in UK affected the recruitment of men wanting to be sperm donors and also affected the attitudes of the practitioners who provide donor sperm treatment?

SUMMARY ANSWER

We have performed fewer IUI and IVF treatments using donor sperm following the change in legislation in April 2005 than before. However, we have seen an overall increase in men wanting to donate their sperm, including a small increase in men from ethnic minorities.

WHAT IS KNOWN ALREADY

Sweden, which removed donor anonymity in 1985, had an initial drop in men wanting to donate and then 10 years later started to have an increase. The Human Fertilisation and Embryology Authority (HFEA) and other studies in the UK have shown an overall downward trend, but have not been able to compare large time scales either side of the change in legislation.

STUDY DESIGN, SIZE, DURATION

This was a retrospective descriptive study that looked at all men who approached the clinic between the years 2000 and 2010, i.e. 5 years either side of the change in legislation (April 2005). Overall, we had 24 men wanting to be donors prior to the rule change and 65 men after the rule change. We also investigated the total number of all treatments with donor sperm, and this included a total of 1004 donor sperm treatments prior to the change in legislation and 403 donor sperm treatments after the change in legislation.

PARTICIPANTS, SETTING, METHODS

The study was set in an NHS IVF clinic in South East London. We compared the indicators of service provision, provider practices and donor attitudes, in the period between April 2000 and March 2005 (Group A) with those between April 2005 and March 2010 (Group B), i.e. 5 years either side of the change in legislation.

MAIN RESULTS

There were 875 IUI treatments and 129 IVF or ICSI treatments in Group A and 325 IUI and 78 IVF/ICSI treatments in Group B with the use of donor sperm, of which, 11.9% (119 out of 1004) in Group A and 39.5% (159 out of 403) in Group B were with donor sperm recruited by our unit. The clinical pregnancy rate per cycle of treatment in Group A was (86 out of 875) 9.8% for IUI and (27 out of 129) 20.9% for IVF/ICSI and in Group B (32/325) 9.8% and (28 out of 78) 35.9%, respectively. There was a sharp yearly fall in donor sperm treatments from 2004. Twenty-four men were screened in Group A, of which 18 (75.0%) were recruited for long-term storage and 12 (50%) were registered as donors with the HFEA when the sperm was used, whereas in Group B, 65 men were screened, 53 (82.0%) were recruited and 24 (36.92%) were registered as donors. Six (24.0%) men in Group A failed in screening because of poor semen analysis when compared with 9 (13.8%) men in Group B. The majority of post-recruitment dropouts were because of loss of follow-up or withdrawal of consent. More donors in Group A were white (92.0 versus 77.0%) and born in UK (92.0 versus 68.0%) when compared with those in Group B. Donors in Group B were more likely to be single (46.0 versus 4.0%) and to have informed their relevant partner of donation (71.0 versus 54.0%) when compared with those in Group A. 83.0% of donors in Group A were heterosexual when compared with 69.0% in Group B. The primary reason for donating in both groups of potential donors was ‘wanting to help’ (46.0% ‘altruistic donors’ in Group A versus 72.0% in Group B). Fewer donors in Group B (37%) had specific restrictions about the use of their sperm when compared with 46.0% in Group A.

LIMITATIONS, REASONS FOR CAUTION

As this was a retrospective study, there is a chance for the introduction of bias.

WIDER IMPLICATIONS OF THE FINDINGS

We have shown that despite no active in-house recruitment procedures, we are managing to recruit more potential sperm donors after the change in UK legislation, and we are able to meet the demand for treatments with in-house recruited donor sperm that is a reassuring finding for donor sperm treatment services in the wider UK.

FUNDING/COMPETING INTERESTS

No external funds were sought for this work. None of the authors have any competing interests to declare.

Source:
http://humrep.oxfordjournals.org/cgi/content/short/28/3/676?rss=1

STUDY QUESTION

Do women who don’t succeed in giving birth after an infertility evaluation have a higher risk of psychiatric disorders compared with women who do?

SUMMARY ANSWER

The results indicated that being unsuccessful in giving birth after an infertility evaluation could be an important risk factor for psychiatric disorders.

WHAT IS KNOWN ALREADY

Several studies have investigated the association between fertility treatment and psychological distress, but the results from these studies show substantial variation and lack of homogeneity that may be due to methodological limitations.

STUDY DESIGN, SIZE AND DURATION

A retrospective cohort study was designed using data from a cohort of 98 320 Danish women evaluated for fertility problems during 1973–2008 and linked to several Danish population-based registries. All women were followed from the date of first infertility evaluation until date of hospitalization for the psychiatric disorder in question, date of emigration, date of death or 31 December 2008, whichever occurred first. Owing to the precise linkage between the infertility cohort and the Danish population-based registries, using the unique Danish personal identification number, virtually no women were lost to follow-up.

PARTICIPANTS/MATERIALS, SETTING AND METHODS

Information on reproductive status for all women in the infertility cohort was obtained by linkage to the Danish Medical Birth Registry. A total of 53 547 (54.5%) women gave birth after the initial infertility evaluation, whereas 44 773 (45.5%) women did not gave birth after the evaluation. To determine psychiatric disorders diagnosed in the women after enrolment in the infertility cohort, the cohort was linked to the Danish Psychiatric Central Registry. A total of 4633 women were hospitalized for a psychiatric disorder. The Cox proportional hazard regression model was applied to estimate hazard ratios (HRs) and corresponding 95% confidence intervals (CIs) for the association between parity status after the initial infertility evaluation and risk of hospitalization for various groups of psychiatric disorders, including ‘all mental disorders’ and six main discharge subgroups labelled: ‘alcohol and intoxicant abuse’, ‘schizophrenia and psychoses’, ‘affective disorders’, ‘anxiety, adjustment and obsessive compulsive disorders’, ‘eating disorder’ and ‘other mental disorders’.

MAIN RESULTS AND THE ROLE OF CHANCE

The incidence rate for all mental disorders was 393 cases per 100 000 person-years among women who did not succeed in giving birth after the infertility evaluation but only 353 cases per 100 000 person-years among women who succeeded in giving birth after the infertility evaluation. Women not giving birth after the infertility evaluation had an increased risk of hospitalization for all mental disorders (HR 1.17, 95% CI 1.11; 1.25), alcohol and intoxicant abuse (HR 2.02, 95% CI 1.69; 2.41), schizophrenia and psychoses (HR 1.46, 95% CI 1.17; 1.82) and other mental disorders (HR 1.42, 95% CI 1.27; 1.58) compared with women who gave birth after the infertility evaluation. In contrast, the risk of affective disorders (HR 0.90, 95% CI 0.81; 0.99) was decreased among women not giving birth after the infertility evaluation. Finally, the risk of anxiety, adjustment and obsessive compulsive disorders (HR 1.07, 95% CI 0.97; 1.17) as well as of eating disorders (HR 1.40, 95% CI 0.88; 2.22) was not significantly affected by parity status after the infertility evaluation.

LIMITATIONS, REASON FOR CAUTION

As only psychiatric conditions warranting hospitalization could be included in the present study, the true incidence of all psychiatric disorders among women with fertility problems is likely to be somewhat underestimated. Furthermore, since detailed information on fertility treatment was not available for all cohort members the association between different modalities of assisted reproductive techniques and risk of psychiatric disorders was not assessed.

WIDER IMPLICATIONS OF THE FINDINGS

Clinicians and other healthcare personnel involved in diagnosis and treatment of women with fertility problems should be aware of the potential risk modification of psychiatric disorders associated with unsuccessful fertility treatment. Hence, our results may point to new aspects of follow-up of women with fertility problems who are unsuccessful in giving birth in order to prevent or identify and treat these possible psychological side effects.

STUDY FUNDING/COMPETING INTEREST(S)

The study was supported by the Danish Cancer Society (award number: 96 222 54). All authors report no conflicts of interest.

Source:
http://humrep.oxfordjournals.org/cgi/content/short/28/3/683?rss=1

STUDY QUESTION

What are the psychometric properties in mainland China of the 30-item Endometriosis Health Profile (EHP-30) translated into simplified Chinese?

SUMMARY ANSWER

The simplified Chinese version of the EHP-30 is a valid, reliable and acceptable tool for the measurement of the health-related quality of life (HRQoL) of women with endometriosis in the context of mainland China.

WHAT IS KNOWN ALREADY

Endometriosis can critically affect women's HRQoL. The EHP-30 is currently the most reliable instrument to measure the HRQoL in women with endometriosis.

STUDY DESIGN, SIZE, DURATION

This cross-sectional study was conducted in a tertiary referral university hospital from February 2012 to August 2012 in Beijing, P. R. China.

PARTICIPANTS/MATERIALS, SETTING, METHODS

The translation and cultural adaptation of the EHP-30 was performed according to accepted guidelines. The study included 336 women with endometriosis. Psychometric evaluation included factor analysis, convergent validity, measurement of internal consistency, item-total correlations and data completeness, descriptive statistics, and the determination of floor and ceiling effects.

MAIN RESULTS AND THE ROLE OF CHANCE

Factor analysis confirmed the validity of the five-factor structure of the EHP-30 core questionnaire, which explained 79.51% of the total variance. The correlations of related subscale scores between EHP-30 and Short Form-36 were all significant. Cronbach's α for internal consistency across each scale ranged 0.89–0.97 for the core questionnaire and 0.80–0.96 for the modular questionnaire. No <97.67% of data completeness was achieved. Floor effects were observed in three scales: self-image (19.64%), children (26.67%) and medical profession (15.19%). No ceiling effects were found. The control and powerlessness scale had the highest median score (54.17) in the core questionnaire, whereas the infertility module (median = 56.25) had the highest score in the modular section.

LIMITATIONS, REASONS FOR CAUTION

The study was conducted in a referral centre for the treatment of endometriosis, thereby leading to overrepresentation of severe symptoms of endometriosis. Furthermore, the test–retest reliability and responsiveness of the questionnaire were not evaluated in this study.

WIDER IMPLICATIONS OF THE FINDINGS

Our study addresses the urgent need for a valid and reliable instrument to measure the HRQoL of female patients with endometriosis in mainland China.

STUDY FUNDING/COMPETING INTEREST(S)

This work was supported by grants to J. Leng from the Key Project for Clinical Faculty Foundation, Ministry of Health, China (2010). None of the authors has any conflict of interest to declare.

Source:
http://humrep.oxfordjournals.org/cgi/content/short/28/3/691?rss=1

STUDY QUESTION

How do apoptosis-related BCL2 and BAX genes, known to regulate death or survival of germ cells in fetal and adult life, and germ-cell-specific VASA protein behave from birth to puberty in the human ovary?

SUMMARY ANSWER

In resting primordial follicles in both infant and pubertal ovaries, BCL2 family members and germ-cell-specific VASA behave as in fetal life. After birth, once follicles leave the resting reserve to enter the growing follicular pool, detection of apoptosis-related genes moves from the germ cell to granulosa cells and VASA expression is lost.

WHAT IS KNOWN ALREADY

In the human ovary, around 85% of the 7 x 10⁶ potential oocytes produced at mid-gestation are lost before birth, an extra 10% before puberty, and loss continues throughout reproductive life until germinal exhaustion of the ovary. Oocyte loss is mainly driven through a balanced expression of BCL2 gene family members. Apoptosis-inducing BAX gene shows a sustained expression throughout fetal and adult life, whereas apoptosis-inhibiting BCL2 is detectable during the proliferative stage of primordial germ cells and oogonia in the fetal ovary and proliferation of granulosa cells in growing follicles in the adult ovary. The germ-cell marker VASA is detectable in the fetal ovary from early oogenesis and is conspicuously expressed in primordial follicles, where in late pregnancy it is associated with the Balbiani's vitelline space. VASA expression is not detectable in the adult ovary.

STUDY DESIGN, SIZE, DURATION

This is a qualitative analysis involving infant/pubertal paraffin-embedded human ovaries screened for apoptosis-related proteins, DNA fragmentation and germ-cell identity.

PARTICIPANTS/MATERIALS, SETTING, METHODS

Ovaries from 13 patients ranging in age from 4 to 16 years, undergoing gynaecological surgical procedures due to benign pathology, were studied. Tissues were fixed in 10% formalin, paraffin-embedded and processed for immunohistochemistry to screen the temporal and cellular localization of germ-cell-specific VASA protein and BCL2 and BAX apoptosis-related proteins. In addition, a terminal deoxynucleotidyl transferase-mediated deoxiuridinetriphosphate nick-end labelling (TUNEL) assay was performed to detect DNA fragmentation. General histology and tissue integrity were assessed by haematoxylin–eosin staining.

MAIN RESULTS AND THE ROLE OF CHANCE

VASA showed a differential pattern of expression; in the resting primordial follicle reserve in infant and pubertal ovaries, it was associated with the Balbiani's body space in the germ cell. VASA remained detectable in primary follicles leaving the resting reserve, but once follicles entered the growing pool it became undetectable. This pattern of VASA expression is the same as in the fetal ovary. BAX was expressed both in the resting primordial reserve and in the pool of growing follicles, whereas BCL2 was detected only in granulosa cells in antral follicles in the growing pool. Apoptosis-related protein expression moved from the germ cell to the somatic stratum when primordial follicles left the resting reserve to enter the pool of growing follicles, irrespective of female age. Most TUNEL-positive cells were detected in the granulosa cells of antral follicles. No TUNEL-positive cells were found in resting primordial follicles.

LIMITATIONS, REASONS FOR CAUTION

The study was limited by the qualitative nature of the immuno-histochemical analysis and the TUNEL assay. The results neither quantify the levels of germ-cell death nor exclude other concurrent cell death mechanisms that could act in the regulation of female germ-cell number.

WIDER IMPLICATIONS OF THE FINDING

This study provides missing knowledge about apoptosis and germ-cell-specific VASA expression in the human ovary between birth and puberty and the participation of BCL2 and BAX genes in the balance between death and survival throughout female germ-line development. Intracellular localization of VASA in resting follicles emerges as a possible marker with prognostic value that needs further investigation, especially in infant patients entering ovarian cryo-preservation programmes. This knowledge will be valuable in optimizing the rescue and clinical use of germ cells to restore fertility in women.

STUDY FUNDING/COMPETING INTEREST(S)

No external funding was obtained for this study. The authors have no conflicts of interest to declare.

TRIAL REGISTRATION NUMBER

Not applicable.

Source:
http://humrep.oxfordjournals.org/cgi/content/short/28/3/698?rss=1

STUDY QUESTION

Does resveratrol counteract age-associated infertility in a mouse model of reproductive aging?

SUMMARY ANSWER

Long-term-oral administration of resveratrol protects against the reduction of fertility with reproductive aging in mice.

WHAT IS KNOWN ALREADY

Loss of oocytes and follicles and reduced oocyte quality contribute to age-associated ovarian aging and infertility. Accumulation of free radicals with age leads to DNA mutations, protein damage, telomere shortening, apoptosis and accelerated ovarian aging. Increasing evidence shows that resveratrol, enriched in certain foods, for example red grapes and wine, has anti-tumor and anti-aging effects on somatic tissues by influencing various signaling pathways, including anti-oxidation, as well as activating Sirt1 and telomerase. We investigated the potential of resveratrol to stave off ovarian aging in the inbred C57/BL6 mouse model.

STUDY DESIGN, SIZE, DURATION

Young C57/BL6 females (aged 2–3 months) were fed with resveratrol added to drinking water at 30 mg/l (providing ~7.0 mg/kg/day) for 6 or 12 months, and the fertility and ovarian functions were compared among mice treated with or without resveratrol, and young mice served as reproductive controls. Experiments were repeated three times, with an average of 25 females randomly allocated to each treatment group for each repeat.

PARTICIPANTS/MATERIALS, SETTING, METHODS

Reproductive performance of female mice was determined by litter size, ovarian follicles and oocyte quantity and quality, and compared with age-matched controls. The impact of resveratrol on telomeres and telomerase activity, and expression of genes associated with cell senescence also was evaluated.

MAIN RESULTS AND THE ROLE OF CHANCE

Young mice fed with resveratrol for 12 months retained the capacity to reproduce, while age-matched controls produced no pups. Consistently, mice fed with resveratrol for 12 months exhibited a larger follicle pool than controls (P < 0.05). Furthermore, telomerase activity, telomere length and age-related gene expression in ovaries of mice fed with resveratrol resembled those of young mice, but differed (P < 0.05) from those of age-matched old mice. Resveratrol improved (P < 0.05) the number and quality of oocytes, as evidenced by spindle morphology and chromosome alignment. Also, resveratrol affected embryo development in vitro in a dose-dependent manner.

LIMITATIONS, REASONS FOR CAUTION

The doses of resveratrol and the experimental conditions used by different research groups have varied considerably, and the dosage influences both the effectiveness and toxicity of resveratrol. Fine-tuning the dosage of resveratrol likely will optimize its anti-aging effects on ovarian function.

WIDER IMPLICATIONS OF THE FINDINGS

Our data provide a proof of principle of the fertility-sparing effect of resveratrol in female mice. Although depletion of the ovarian reserve of high-quality oocytes also contributes to increased infertility with reproductive aging in women, the data obtained using a mouse model may not extrapolate directly to human reproduction, and more extensive research is needed if any clinic trials are to be attempted.

STUDY FUNDING/COMPETING INTEREST(S)

This work was supported by MOST of China National Basic Research Program (grant number: 2010CB94500 and 2012CB911200). The authors have no competing interests to declare.

Source:
http://humrep.oxfordjournals.org/cgi/content/short/28/3/707?rss=1