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Category Archives: Human Reproduction

Motile sperm organelle morphology evaluation-selected globozoospermic human sperm with an acrosomal bud exhibits novel patterns and higher levels of phospholipase C zeta

STUDY QUESTION

Does motile sperm organelle morphology examination (MSOME) affect levels and localization patterns of the oocyte activation factor phospholipase C zeta (PLC) in globozoospermic sperm with and without an acrosomal bud?

SUMMARY ANSWER

MSOME identified round-headed globozoospermic sperm with increased levels of PLC relative to sperm from the same sample that did not undergo MSOME, and identified novel patterns of PLC localization in sperm exhibiting an acrosomal bud.

WHAT IS KNOWN ALREADY

Absence or reduction in the level of PLC in the sperm head, abnormal localization patterning, or defective functional ability as a result of PLC gene mutation, have been linked to certain types of human male factor infertility in which oocyte activation is deficient. It has been determined that a subpopulation of sperm (1%) from a patient exhibiting 100% globozoospermia presented with an acrosome bud upon MSOME. A cycle of intracytoplasmic morphologically selected sperm injection, carried out with sperm exhibiting an acrosomal bud led to pregnancy and birth of a healthy baby boy, without the use of assisted oocyte activation (AOA).

STUDY DESIGN, SIZE, DURATION

Immunofluorescent analysis of PLC in globozoospermic sperm from three patients, before and after MSOME.

PARTICIPANTS/MATERIALS, SETTING, METHODS

Quantitative immunofluorescence was used to investigate PLC levels and localization patterns in individual sperm (n = 1 patient) identified by MSOME and isolated by micromanipulation, and presenting with and without the acrosomal bud. A secondary aim was to investigate levels and localization patterns of PLC in sperm before and after MSOME from two other globozoospermic men.

MAIN RESULTS AND THE ROLE OF CHANCE

Non-globozoospermic control sperm exhibited characteristic localization patterns of PLC immunofluorescence. Completely round-headed globozoospermic sperm from patients 1–3 were either devoid of PLC immunofluorescence, or exhibited an abnormal, punctate, pattern of PLC localization. PLC immunofluorescence in sperm exhibiting an acrosomal bud was observed in the midpiece with varying fluorescent intensity and was detected in 28.5% of such sperm. The majority of sperm with an acrosomal bud (43.0%) exhibited punctate patterns of PLC localization within the sperm head. A further 28.5% of sperm exhibited PLC in both the head and the midpiece. Total levels of PLC, and the proportions of sperm exhibiting PLC immunoreactivity, showed significant variance (P ≤ 0.05) amongst control [45.8 arbitrary units (a.u.) and 95.7%, respectively], non-MSOME-selected (25.9 a.u. and 46.1%, respectively) and MSOME-selected globozoospermic sperm (33.4 a.u. and 65.0%, respectively). Total levels of PLC immunofluorescence, and proportions of sperm exhibiting PLC immunoreactivity, in control sperm was significantly higher (P≤ 0.05) compared with non-MSOME-selected sperm, but not significantly different from MSOME-selected sperm.

LIMITATIONS, REASONS FOR CAUTION

The low numbers of sperm analysed may not be ideal for conclusive statistical analysis. Evaluation of the effects of MSOME on morphologically normal sperm would confirm conclusions.

WIDER IMPLICATIONS OF THE FINDINGS

The present findings provide hope for the future treatment of globozoospermia without the need for AOA, and provide further evidence for the clinical application of PLC as a therapeutic and prognostic tool.

STUDY FUNDING/COMPETING INTEREST(S)

The research described herein was funded by the Nuffield Department of Obstetrics and Gynaecology, University of Oxford. The authors report no conflict of interest.

Source:
http://humrep.oxfordjournals.org/cgi/content/short/27/11/3150?rss=1

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Effect of smoking on the functional aspects of sperm and seminal plasma protein profiles in patients with varicocele

STUDY QUESTION

What are the effects of smoking on the functional aspects of the sperm, the levels of lipid peroxidation and the protein profile of seminal plasma in patients with varicocele?

SUMMARY ANSWER

In men with varicocele, smoking is associated with altered semen quality, decreased sperm functional integrity and seminal oxidative stress. Alterations in seminal plasma protein profiles are also present and may explain the altered semen phenotype.

WHAT IS KNOWN ALREADY

Varicocele is a major cause of male infertility. It reduces testicular blood renewal with a consequent accumulation of toxic substances. Thus, it can potentiate the toxic effects of environmental exposure to genotoxic substances such as those found in cigarette smoke.

STUDY DESIGN, SIZE AND DURATION

A cross-sectional study was performed in 110 patients presenting with variococele to the Human Reproduction Section of the Sao Paulo Federal University (2006–2010). The patients were divided into a control group of non-smokers, a moderate smokers group and a heavy smokers group.

PARTICIPANTS/MATERIALS, SETTING AND METHODS

Semen parameters were analysed by standard methods. Sperm DNA integrity and mitochondrial activity were assessed by Comet assays and by 3,3'-diaminobenzidine deposition, respectively. The level of lipid peroxidation in semen was determined by malondialdehyde quantification. Proteomic studies were performed by 2D-electrophoresis and mass spectrometry.

MAIN RESULTS AND THE ROLE OF CHANCE

Both groups of smokers showed reduced semen quality in comparison with the control group. In the groups of smokers, sperm DNA integrity and mitochondrial activity were also decreased and lipid peroxidation levels were increased. Proteomic analyses revealed 20 proteins differentially expressed between the study groups.

LIMITATIONS AND REASONS FOR CAUTION

A study including smokers without varicocele is still warranted as these results apply only to smokers who present varicocele.

WIDER IMPLICATIONS OF THE FINDINGS

Patients with varicocele who are exposed to tobacco smoking present more important alterations to semen quality and sperm functional integrity and show changes in the seminal plasma proteome. This suggests testicular, and possibly systemic, adverse effects of smoking.

STUDY FUNDING/COMPETING INTEREST(S)

Funding for the study was provided by Fundação de Amparo à Pesquisa do Estado de São Paulo (Fapesp) (2007/59423-7) and by the Division of Urology, Human Reproduction Section at the São Paulo Federal University.

Source:
http://humrep.oxfordjournals.org/cgi/content/short/27/11/3140?rss=1

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Monitoring fertility (semen analysis) by cancer survivors who banked sperm prior to cancer treatment

STUDY QUESTION

What medical and psychological variables predict why men with banked sperm do not return for semen analysis after their cancer treatment has ended?

SUMMARY ANSWER

Men who decline the offer of semen analysis are less likely to have reported adverse side effects during cancer treatment, and have a more negative experience of banking sperm and a more negative attitude towards disposal of their stored semen than those who attend.

WHAT IS KNOWN ALREADY

Previous authors have noted that male cancer survivors seem reluctant to have their fertility tested after their treatment has ended. Moreover, the utilization rates of banked sperm are very low (<10%) and the majority of samples are kept for many years without being used.

STUDY DESIGN, SIZE AND DURATION

A cross-sectional study of 499 cancer survivors who were sent a questionnaire about their views on sperm banking, fertility and post-treatment semen analysis between April 2008 and December 2010.

PARTICIPANTS AND SETTING

Men (aged 18–55 years) who had banked sperm in Sheffield and Nottingham (UK) prior to gonadotoxic treatment for cancer more than 5 years previously.

MAIN RESULTS AND THE ROLE OF CHANCE

Completed questionnaires were received from 193 men (38.7% response rate) whose samples had been banked for 9.18 ± 3.70 years (range = 4.94–26.21) and whose current age was 35.08 ± 7.08 years (range = 21.58–54.34; mean ± SD). One-third (35.8%) had never attended for semen analysis. In multivariate analysis, the odds of not attending for semen analysis were significantly greater among men who did not experience adverse treatment side effects [odds ratio (OR) = 5.72, 95% confidence interval (CI) = 2.10–15.56], who reported a more negative experience of banking sperm (OR = 1.82, 95% CI = 1.17–2.82) and a more negative attitude to disposal of their stored semen (OR = 1.56, 95% CI = 1.01–2.42).

LIMITATIONS AND REASONS FOR CAUTION

Only 38.7% of those eligible agreed to take part. We do not know the characteristics of men who declined to take part, if they agreed to attend semen analysis without completing the questionnaire or whether they had chosen to have semen analysis performed elsewhere (e.g. private sector). Some of the measures used (e.g. experience of banking sperm) relied on men's recall of events many years previously.

WIDER IMPLICATIONS OF THE FINDINGS

New strategies are required to encourage these men to engage with fertility monitoring programmes if sperm banks are to be used cost-effectively and men are to be given appropriate fertility advice.

STUDY FUNDING AND COMPETING INTERESTS

This paper was supported by funding from Cancer Research-UK to C.E., A.A.P. and R.R. (C481/A8141). The views expressed are those of the authors. No competing interests declared.

Source:
http://humrep.oxfordjournals.org/cgi/content/short/27/11/3132?rss=1

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Fertilization rates are improved after IVF if the corona radiata is left intact in vitrified-warmed human oocytes

BACKGROUND

Before human MII oocytes are vitrified they are usually denuded from their cumulus cells. In this study we wanted to investigate the effects of an intact corona radiata on the vitrification and fertilization of human oocytes.

METHODS

The study comprised two different parts. In Part 1, 36 MII stage oocytes, from 6 patients, were randomly assigned into a control group, a group of vitrified-warmed oocytes without a corona radiata and a group of vitrified-warmed oocytes with an intact corona radiata. In each group of 12, 6 oocytes were used for evaluation of the zona pellucida solubility (hardening) and another 6 oocytes were used for the analysis of their ultrastructure. In addition, six polyspermically fertilized oocytes were used as positive controls for zona pellucida hardening. In Part 2, 16 patients in total produced 107 fresh and 98 vitrified-warmed oocytes, with or without an intact corona radiata. All oocytes were fertilized via conventional IVF and embryos were transferred according to our standard ET routines. The oocyte survival and fertilization rates, embryo quality and pregnancy and implantation rates were evaluated.

RESULTS

There were no differences in oocyte survival, zona pellucida solubility (hardening) or the number of cortical granules between the vitrified-warmed and fresh oocytes. There were also no differences in the zona pellucida solubility and the number of cortical granules between vitrified-warmed oocytes with or without an intact corona radiata. However, the oocytes with an intact corona radiata had a higher fertilization rate after conventional IVF insemination. No differences were seen in the survival and cleavage rates, the percentage of high-quality embryos or the clinical outcome.

CONCLUSIONS

Zona hardening and ultrastructural damage do not seem to occur in vitrified human oocytes. An intact corona radiata in vitrified-warmed oocytes retains their fertilization capacity in conventional IVF, but does not improve the embryo quality. Poor fertilizing capacities of vitrified-warmed oocytes without an intact corona radiata seem to have been due to the complete removal of the cumulus cells.

Source:
http://humrep.oxfordjournals.org/cgi/content/short/27/11/3208?rss=1

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The association between severe obesity and characteristics of failed fertilized oocytes

STUDY QUESTION

Is the cytoskeletal and chromosomal organization of failed fertilized oocytes from severely obese patients (BMI ≥ 35 kg/m2) altered compared with that in patients with normal BMI (BMI 18.5–24.9 kg/m2)?

SUMMARY ANSWER

Compared with normal BMI patients, severe obesity was associated with a greater prevalence of spindle anomalies and non-aligned chromosomes in failed fertilized oocytes.

WHAT IS KNOWN AND WHAT THIS PAPER ADDS

Obesity is associated with poor reproductive outcomes, but little is known regarding the underlying mechanisms. To address potential mechanisms, our study compared the cytoskeletal and chromosome organization in failed fertilized oocytes from severely obese and normal BMI patients.

DESIGN

The study population was drawn from IVF patients treated in a hospital-based infertility clinic between February 2010 and July 2011. The prevalence of meiotic spindle and chromosome alignment anomalies in failed fertilized oocytes from patients with severe obesity (i.e. Class II and III; BMI 35.0–50.1 kg/m2) was compared with those from patients with normal BMI (BMI 18.5–24.9 kg/m2). Oocytes were fixed and then labeled for tubulin, actin and chromatin. Spindle number and integrity, as well as chromosome alignment, were assessed using immunofluorescence microscopy and, in some cases, confocal microscopy. Generalized estimating equations were applied, which account for the correlation among oocytes from the same patient to estimate odds ratio (OR), 95% confidence intervals (CIs) and two-sided Wald P-values. Models were adjusted for continuous age at cycle start, cycle type (IVF or ICSI) and polycystic ovarian syndrome (PCOS) a priori.

PARTICIPANTS AND SETTING

University-affiliated infertility clinic. A total of 276 oocytes that failed to fertilize from 137 patients were evaluated: 105 oocytes from severely obese women (n = 47) and 171 oocytes from normal BMI patients (n = 90).

MAIN RESULTS AND THE ROLE OF CHANCE

(i) Significantly more oocytes from the severely obese group exhibited two spindles compared with those from the normal BMI group (58.9 versus 35.1%; OR = 2.68, CI = 1.39–5.15, P-value = 0.003).

(ii) Among oocytes with a single spindle, those from severely obese patients showed a significantly higher prevalence of disarranged spindles with non-aligned chromosomes compared with those from normal BMI patients (28.6 versus 8.6%; OR = 4.58, CI = 1.05–19.86, P-value = 0.04).

BIAS, CONFOUNDING AND OTHER REASONS FOR CAUTION

Inclusion of only failed fertilized oocytes, small sample size, unknown factors such as non-PCOS comorbidity.

GENERALIZABILITY TO OTHER POPULATIONS

For this study, by design, it is unclear whether the findings are generalizable to successfully fertilized oocytes, and whether this oocyte-level influence of obesity is generalizable to infertile women who do not undergo stimulation or, more broadly, to spontaneous conceptions in fertile women.

STUDY FUNDING/COMPETING INTEREST(S)

none.

TRIAL REGISTRATION NUMBER

n/a.

Source:
http://humrep.oxfordjournals.org/cgi/content/short/27/11/3198?rss=1

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Common chromosomal imbalances and stemness-related protein expression markers in endometriotic lesions from different anatomical sites: the potential role of stem cells

BACKGROUND

Endometriosis is a multifactorial gynecological disease characterized by the presence of functional endometrium-like tissue in ectopic sites. Several studies have focused on elucidating the immunological, endocrine, environmental and genetic factors involved in endometriosis. However, its pathogenesis is still unclear.

METHODS

High-resolution comparative genomic hybridization was applied to screen for genomic imbalances in laser microdissected stromal and epithelial cells from 20 endometriotic lesions and three samples of eutopic endometrium derived from eight patients. The expression of seven stemness-related markers (CD9, CD13, CD24, CD34, CD133, CD117/c-Kit and Oct-4) in endometrial tissue samples was evaluated by immunohistochemistry.

RESULTS

Samples of eutopic endometrium showed normal genomic profiles. In ectopic tissues, an average of 68 genomic imbalances was detected per sample. DNA losses were more frequently detected and involved mainly 3p, 5q, 7p, 9p, 11q, 16q, 18q and 19q. Many of the genomic imbalances detected were common to endometriotic stroma and epithelia and also among different endometriotic sites from the same patient. These findings suggested a clonal origin of the endometriotic cells and the putative involvement of stem cells. Positive immunostaining for CD9, CD34, c-Kit and Oct-4 markers was detected in isolated epithelial and/or stromal cells in eutopic and ectopic endometrium in the majority of cases.

CONCLUSIONS

The presence of shared genomic alterations in stromal and epithelial cells from different anatomical sites of the same patient and the expression of stemness-related markers suggested that endometriosis arises as a clonal proliferation with the putative involvement of stem cells.

Source:
http://humrep.oxfordjournals.org/cgi/content/short/27/11/3187?rss=1

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