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Category Archives: Human Reproduction
The prevalence and impact of fibroids and their treatment on the outcome of pregnancy in women with recurrent miscarriage
BACKGROUND
Although uterine fibroids have been associated with spontaneous miscarriage, to our knowledge there have been no studies in the literature assessing their role in the recurrent miscarriage (RM) population. The aims of this study are to examine the impact of different types of fibroids on the pregnancy outcome of women with RM and to investigate to what extent resection of fibroids distorting the uterine cavity affects the outcome of a future pregnancy.
METHODS
The study analysed retrospective and prospective data from a large tertiary referral RM clinic. Couples were investigated as per an established protocol. Fibroids were diagnosed using combined transvaginal ultrasound and hysterosalpingography. Fibroids distorting the uterine cavity were resected via hysteroscopy. Two study groups were subsequently examined: women with cavity-distorting fibroids who underwent surgery (n =25) and women with fibroids not distorting the cavity who did not undergo any intervention (n =54). The latter was compared with a control group of women with unexplained RM (n =285).
RESULTS
The prevalence of fibroids was found to be 8.2% (79/966). In total, 264 pregnancies of women with fibroids and 936 pregnancies of women with unexplained RM were analysed. Women with intracavitary distortion and undergoing myomectomy significantly reduced their mid-trimester miscarriage rates in subsequent pregnancies from 21.7 to 0% (P< 0.01). This translated to an increase in the live birth rate from 23.3 to 52.0% (P< 0.05). Women with fibroids not distorting the cavity behaved similarly to women with unexplained RM achieving a 70.4% live birth rate in their subsequent pregnancies without any intervention.
CONCLUSIONS
Fibroids are associated with increased mid-trimester losses amongst women with RM. Resection of fibroids distorting the uterine cavity can eliminate the mid-trimester losses and double the live birth rate in subsequent pregnancies. Women with fibroids not distorting the uterine cavity can achieve high live birth rates without intervention.
Posted in Human Reproduction
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Evaluation of correct endogenous reactive oxygen species content for human sperm capacitation and involvement of the NADPH oxidase system
BACKGROUND
Generation of controlled amounts of reactive oxygen species (ROS) and phosphorylation of protein tyrosine residues (Tyr) are two closely related changes involved in sperm capacitation. This study investigated the effect of altered endogenous ROS production on Tyr-phosphorylation (Tyr-P), acrosome reaction (AR) and cell viability during sperm capacitation. The possible origin of the altered ROS production was also evaluated by apocynin (APO) or oligomycin (Oligo) addition.
METHODS
A total of 63 samples of purified sperm were analysed for ROS production by enhanced chemiluminescence, Tyr-P pattern by immunocytochemistry, and AR and viability by fluorochrome fluorescein isothiocyanate (FITC)-labelled peanut (Arachis hypogaea) agglutinin and propidium iodide positivity, respectively.
RESULTS
Samples were divided into four categories depending on the ability of sperm to produce ROS, expressed as Relative Luminescence Units (RLU), in capacitating conditions: low ROS production (LRP), range about 0.0–0.05 RLU; normal (NRP), 0.05–0.1 RLU; high (HRP), 0.1–0.4 RLU; very high (VHRP) 0.4–2.0 RLU. In NRP sperm heads, capacitation induced Tyr-P in 87.9 ± 4.3%, and the AR occurred in 62.5 ± 5.4% of cells; in LRP, HRP and VHRP Tyr-P labelling rarely spread over the head, acrosome-reacted cells only accounted for a small number of sperm, and the non-viable cells (NVC) were increased. The addition of APO, but not Oligo, drastically decreased ROS production in analysed samples.
CONCLUSIONS
This study proposes the optimal threshold for endogenous ROS production for correct sperm viability and functioning, and indicates the direct involvement of APO-sensitive NADPH oxidase in ROS production.
Posted in Human Reproduction
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Exogenous pyruvate accelerates glycolysis and promotes capacitation in human spermatozoa
BACKGROUND
There has been an ongoing debate in the reproductive field about whether mammalian spermatozoa rely on glycolysis, oxidative phosphorylation or both for their energy production. Recent studies have proposed that human spermatozoa depend mainly on glucose for motility and fertilization but the mechanism behind an efficient glycolysis in human spermatozoa is not well understood. Here, we demonstrate how human spermatozoa utilize exogenous pyruvate to enhance glycolytic ATP production, motility, hyperactivation and capacitation, events that are crucial for male fertility.
METHODS
Purified human spermatozoa from healthy donors were incubated under capacitating conditions (including albumin, bicarbonate and glucose) and tested for changes in ATP levels, motility, hyperactivation and tyrosine phosphorylation after treatment with pyruvate. The experiments were repeated in the presence of sodium cyanide in order to assess the contribution from mitochondrial respiration. The metabolism of 13C labeled glucose and pyruvate was traced by a combination of liquid chromatography and mass spectrometry.
RESULTS
The treatment of human spermatozoa with exogenous pyruvate increased intracellular ATP levels, progressive motility and hyperactivation by 56, 21 and 130%, respectively. In addition, added pyruvate induced a significant increase in tyrosine phosphorylation levels. Blocking of the electron transport chain did not markedly affect the results, indicating that the mechanism is independent of oxidative phosphorylation. However, the observed effects could be counteracted by oxamate, an inhibitor of lactate dehydrogenase (LDH). Metabolic tracing experiments revealed that the observed rise in ATP concentration resulted from an enhanced glycolytic flux, which was increased by more than 50% in the presence of exogenous pyruvate. Moreover, all consumed 13C labeled pyruvate added was converted to lactate rather than oxidized in the tricarboxylic acid cycle.
CONCLUSIONS
Human spermatozoa seem to rely mainly, if not entirely, on glycolysis as the source of ATP fueling the energy-demanding processes of motility and capacitation. The efficient glycolysis is dependent on exogenous pyruvate, which indirectly feeds the accelerated glycolysis with NAD+ through the LDH-mediated conversion of pyruvate to lactate. Pyruvate is present in the human female reproductive tract at concentrations in accordance with our results. As seen in other mammals, the motility and fertility of human spermatozoa seem to be dictated by the available energy substrates present in the conspecific female.
Posted in Human Reproduction
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Intra-individual variation of the sperm chromatin structure assay DNA fragmentation index in men from infertile couples
BACKGROUND
The sperm chromatin structure assay (SCSA) is a valuable tool for prediction of fertility in vivo, with DNA fragmentation index (DFI) of 30% as a clinically useful cut-off level. Previous studies on fertile men have shown a high level of repeatability, with an intra-individual variability in DFI of ~10%. However, conflicting data on how much the DFI fluctuates within individuals exist. The aim of the present study was to investigate the intra-individual variation of DFI in order to further evaluate the clinical use of SCSA.
METHODS
Among 2409 consecutive men under infertility investigation, repeated SCSA analyses were performed on 616 samples from men between 18 and 66 years of age. The coefficient of variation (CV) for DFI was calculated. For each patient, we also analyzed whether the DFI value in tests I and II switched the category from <30 to >30%, or vice versa.
RESULTS
Mean CV for DFI for men with at least two SCSA analyses within a 30-month period was 30.1% (SD 21.5). Compared with the first test, 85% (95% confidence interval: 82–87%) of the men remained on the same side of the cut-off point of 30%.
CONCLUSIONS
Despite showing a high intra-individual CV for DFI, 85% of the men from infertile couples did not change category between tests, with respect to the cut-off level of 30%. Thus, using the previously established DFI cut-off value of 30%, a single SCSA analysis has a high predictive value for assessing fertility in vivo.
Posted in Human Reproduction
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Chromosome X-encoded cancer/testis antigens show distinctive expression patterns in developing gonads and in testicular seminoma
BACKGROUND
Cancer/testis (CT) antigens are cancer antigens normally expressed in adult testicular germ cells. The expression of chromosome X-encoded CT antigens (CT-X antigens) in human fetal gonads and in testicular seminomas was examined.
METHODS
The expression of 10 CT-X antigens (MAGEA, NY-ESO-1, GAGE, CT7/MAGEC1, CT10/MAGEC2, CT45, SAGE1, SSX2, NXF2 and SPANX) was studied immunohistochemically.
RESULTS
In adult human testis, SPANX is expressed in late spermatids and spermatozoa, whereas all other CT-X antigens are predominantly expressed in spermatogonia or primary spermatocytes. All CT-X antigens except SPANX are expressed in human fetal germ cells. CT-X-positive germ cells appear as early as 13 weeks after gestation, increase with age and reach a plateau at around 22 weeks. In the fetal ovary, CT-X-positive oogonia are most abundant at around 24 weeks and sharply decrease subsequently. CT-X antigens are almost exclusively expressed in OCT3/4-negative gonocytes and their expression appears to coincide with the loss of pluripotency. Spermatocytic seminoma, a neoplasm derived from adult pre-meiotic germ cells, showed uniform expression of all CT-X antigens except SPANX. In contrast, most seminomas (>80%) express CT7, CT45, GAGE and CT10 but express MAGEA, NXF2 and NY-ESO-1 at lower frequency, and very rarely express SSX2 and SAGE1.
CONCLUSIONS
Most CT-X antigens are expressed in human fetal germ cells after they have lost stem cell characteristics, with predominant expression in pre-meiotic germ cells. Spermatocytic seminomas showed expression of all CT-X antigens except SPANX, whereas classical seminomas only express some CT-X antigens, reflecting their different origins from adult versus fetal germ cells.
Posted in Human Reproduction
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Separating spermatogonia from cancer cells in contaminated prepubertal primate testis cell suspensions
BACKGROUND
Chemotherapy and radiation treatments for cancer and other diseases can cause male infertility. There are currently no options to preserve the fertility of prepubertal boys who are not yet making sperm. Cryopreservation of spermatogonial stem cells (SSCs, obtained via testicular biopsy) followed by autologous transplantation back into the testes at a later date may restore fertility in these patients. However, this approach carries an inherent risk of reintroducing cancer.
METHODS
To address this aspect of SSC transplantation safety, prepubertal non-human primate testis cell suspensions were inoculated with MOLT4 T-lymphoblastic leukemia cells and subsequently sorted for cell surface markers CD90 (THY-1) and CD45.
RESULTS
Cancer cells segregated to the CD90–/CD45+ fraction and produced tumors in nude mice. Nearly all sorted DEAD box polypeptide 4-positive (VASA+) spermatogonia segregated to the CD90+/CD45– fraction. In a preliminary experiment, a purity check of the sorted putative stem cell fraction (CD90+/CD45–) revealed 0.1% contamination with cancer cells, which was sufficient to produce tumors in nude mice. We hypothesized that the contamination resulted from mis-sorting due to cell clumping and employed singlet discrimination (SD) in four subsequent experiments. Purity checks revealed no cancer cell contamination in the CD90+/CD45– fraction from three of the four SD replicates and these fractions produced no tumors when transplanted into nude mouse testes.
CONCLUSIONS
We conclude that spermatogonia can be separated from contaminating malignant cells by fluorescence-activated cell sorting prior to SSC transplantation and that post-sorting purity checks are required to confirm elimination of malignant cells.
Posted in Human Reproduction
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