Steven Fleming

Prospective controlled trial of an electrophoretic method of sperm preparation for assisted reproduction: comparison with density gradient centrifugation

BACKGROUND A membrane-based electrophoretic filtration system, known as the Cell Sorter-10 (CS-10), that preferentially isolates spermatozoa with very low levels of DNA damage has recently been developed. However, it remains to be proven whether spermatozoa prepared in this way are capable of achieving fertilization in assisted conception. Therefore, this clinical trial was designed to answer this question. METHODS A split-sample split-cohort study design was employed to control for differences in semen and oocyte quality between 28 couples undergoing either intracytoplasmic sperm injection (ICSI) or IVF in this clinical trial. Each semen sample was split between preparation using the CS-10 and preparation by standard density gradient centrifugation (DGC) and each cohort of oocytes was split for insemination using either CS-10 (n = 197) or DGC (n = 195) prepared spermatozoa. RESULTS Both methods of sperm preparation yielded comparable rates of sperm recovery, motility and DNA fragmentation. There was no significant difference between the ability of CS-10 and DGC prepared spermatozoa to produce fertilization (62.4% versus 63.6%), cleavage (99.0% versus 88.5%) and high-quality embryos (27.4% versus 26.1%). CONCLUSIONS This pilot study demonstrates that membrane-based electrophoresis is as effective as DGC in preparing sperm for IVF and ICSI, although it takes only a fraction of the time.

The effect of repeated freezing and thawing on human sperm DNA fragmentation

OBJECTIVE To investigate the effects of repeated freezing and thawing on human sperm motility, vitality, and DNA integrity. DESIGN A prospective clinical study. SETTING Tertiary care fertility clinic. PATIENT(S) Twenty men presenting for infertility investigations. INTERVENTION(S) Each sample was subjected to three cycles of freezing and thawing both with and without washing steps and the addition of fresh cryoprotectant between each cycle. MAIN OUTCOME MEASURE(S) Percentage sperm DNA fragmentation, motility, and vitality before and following repeated freezing and thawing. RESULT(S) The percentage sperm DNA fragmentation rose significantly following each freeze-thaw cycle; however, samples that were not washed and to which fresh cryoprotectant was not added after each cycle fared significantly better than their washed counterparts. Both motility and vitality decreased steadily following each cycle but cell survival was significantly greater in the unwashed samples. CONCLUSION(S) In terms of the level of sperm DNA fragmentation, up to three cycles of freezing and thawing can be performed with a level of risk comparable to that following a single cycle of freezing and thawing. This is provided that samples are refrozen in their original cryoprotectant and not washed or altered in any way in between, and provided that they are separated by density gradient centrifugation or swim-up before use in assisted reproduction technology.

Exogenous platelet-activating factor stimulates cell proliferation in mouse pre-implantation embryos prior to the fourth cell cycle and shows isoform-specific stimulatory effects.

Mouse embryos secrete molecules homologous to platelet-activating factor (PAF), termed embryo-derived PAF (EPAF), which act in an autocrine/paracrine fashion to stimulate embryonic development in vitro. Mouse EPAF is thought to consist predominantly of hexadecyl (C16) and octadecyl (C18) PAF-like components. Mouse pre-implantation embryos cultured with exogenous PAF from the early cleavage stages exhibit increased blastocyst cell numbers and rates of mitosis around the 8-cell stage. We investigated whether exogenous PAF could specifically stimulate embryonic cell proliferation prior to the blastocyst stage in the mouse and also compared the biological activities of the C16 and C18 PAF isoforms as follows. Embryos were cultured for either 24 h or 120 h from the 2-cell stage and their total cell numbers were determined or their development assessed in terms of their incidence of successful zona-hatching respectively. In each case, embryos were cultured in unsupplemented medium or in medium supplemented with either C16 or C18 PAF (0.5 microM). Compared with controls, culture with C16 PAF produced a significant stimulation of mean total per number per embryo and a significant increase in the incidence of successful zona-hatching, whilst culture with C18 PAF had no significant effect. We then cultured 1-cell zygotes for 48 h in unsupplemented medium or medium supplemented with either an equimolar mixture of C16 and C18 PF or with either C16 or C18 PF alone (each at 0.2 microM). Embryos were also scored for cell number at 4 h and 30 h of culture. Although no significant effect on mean cell number per embryo was seen following 4 h or 30 h of culture with a mixed C16/C18 PAF preparation, culture for 48 h with a mixed C16/C18 PAF preparation or with C16 PAF alone produced a significant increase in mean cell number per embryo compared with controls - an effect that is likely to be receptor-mediated, since culture with an equivalent concentration of C18 PAF had no significant effect compared with controls. We have demonstrated that mouse zygotes/embryos can respond in a specific manner to exogenous hexadecyl PAF in terms of increased rates of cell proliferation prior to cavitation, and must be capable of doing so at some time between the first and third, and also between the second and fourth, cell cycles. Such embryos presumably express one or more classes of functional PAF-receptor molecule during this period (i.e. as early as during the 1-, 2- or 4-cell stages). We have also demonstrated that embryonic response to exogenous PAF is significantly isoform-specific, which may reflect differences between the two isoforms either in affinity for binding to putative embryonic PAF-receptor molecules or in their ability to elicit a stimulatory response following binding. This observation calls into question the use of preparations containing a mixture of hexadecyl and octadecyl PAF isoforms, particularly in dose-response studies, in the mouse.

The DNA integrity of cryopreserved spermatozoa separated for use in assisted reproductive technology is unaffected by the type of cryoprotectant used but is related to the DNA integrity of the fresh separated preparation

OBJECTIVE To investigate and compare seven different commercially available cryoprotectant media in terms of the DNA integrity of spermatozoa recovered after cryopreservation and separation using density gradient centrifugation (DGC). DESIGN A prospective clinical study. SETTING Tertiary care fertility clinic. PATIENT(S) Three hundred twenty men presenting for infertility investigations. INTERVENTION(S) Each sample was randomly assigned to one of seven commercially available cryoprotectants or to no cryoprotectant. MAIN OUTCOME MEASURE(S) Percentage sperm DNA fragmentation after cryopreservation and preparation using DGC. RESULT(S) The mean percentage fragmentation was significantly higher post-thaw and post-DGC; however, some patients (26.3%) demonstrated a lower percentage fragmentation post-thaw. No single cryoprotectant was identified as the best at preserving DNA integrity. The difference in fragmentation after thawing and DGC was found to be highly dependent on the prefreeze fragmentation. Motility was also significantly correlated with the difference in fragmentation post-thaw (r = -0.161). CONCLUSION(S) Neither the presence nor type of cryoprotectant affects the DNA integrity of spermatozoa after cryopreservation and DGC. Individuals with lower prefreeze fragmentation in DGC-prepared spermatozoa have larger increases in fragmentation and are less likely to exhibit lower levels of fragmentation post-thaw. The reverse effect observed in those with higher prefreeze fragmentation gives rise to a possible novel method of reducing fragmentation in sperm used for assisted reproductive technology treatment cycles without the need for testicular sperm retrievals.

Embryonic Platelet-Activating Factor: Temporal Expression of the Ligand and Receptor

AbstractPurpose: Preimplantation embryos synthesize platelet-activating factor (PAF) and this embryo-derived PAF is required for development. PAFs signal transduction is receptor-mediated and PAF-receptor mRNA is present in early embryos. The study objective was to determine the relationship between PAF production and PAF-receptor mRNA expression levels in mouse preimplantation embryos. Methods: Embryo-derived PAF levels were determined by radioimmunoassay. Embryonic PAF-receptor mRNA levels were determined by semiquantitated reverse transcription-polymerase chain reaction. Results: Embryonic-PAF increased as time progressed at a relatively constant rate (1.4–1.9×) except between the eight- and morula-cell stages where levels increased sevenfold. Embryonic PAF-receptor expression was highest at the two-cell stage and decreased steadily until the morula stage before increasing again. Regression analysis of embryo-derived PAF on PAF-receptor expression does not demonstrate a significant relationship. Conclusions: PAF-receptor expression (mRNA) levels decrease, while embryo-derived PAF levels decrease, as the preimplantation embryo develops. Embryonic-PAF and PAF-receptor mRNA expression do not appear related. Therefore, embryonic-PAF does not appear to regulate expression of its own receptor in vitro. The data provide a clue to the complicated cell signaling system involving PAF production and receptor expression that may help our understanding of developmental events.

Ubiquitin is associated with the survival of ectopic stromal cells in endometriosis

BackgroundEndometriosis is a condition that affects women of reproductive age, where the glandular and/or stromal tissues from the eutopic endometrium implant in ectopic locations. It is well established that the survival of ectopic implants is due to lower levels of apoptosis, but no consensus exists as to which pathway/s this is mediated by. The ubiquitin protein shares a similar sequence homology to an anti-apoptotic protein called BAG-1 and is expressed in the normal endometrium. Currently, no studies have been conducted to determine ubiquitin expression and its possible anti-apoptotic effects in endometriosis.MethodsArchived endometrial tissues from endometriosis patients and women undergoing laparoscopic diagnosis (controls) from January 2000 to July 2003 at Westmead Hospital were examined, where 14 cases of endometriosis and 55 controls were included in the study.ResultsBoth the ubiquitin protein and apoptosis were expressed in both glandular and stromal cells throughout the menstrual cycle of the eutopic endometrium, in which ubiquitin exhibited a cyclic expression, reaching a peak in late proliferative phase. In contrast, ubiquitin was predominantly expressed in cells of stromal origin in endometriosis, was no longer regulated by a cyclic pattern and was associated with an aberrant level of cell survival.ConclusionsFor the first time, this study shows that ubiquitin is expressed in endometriotic cells and may contribute to a reduced sensitivity of ectopic endometrial tissue to apoptosis. These findings also suggest that stromal cells contribute differentially to the development of ectopic endometrial tissue.

Computer image sperm selection as a novel approach to subzonal insemination in the human

Utilizing real-time computer image analysis, individual spermatozoa were selected using microaspiration. Selection criteria were based on potential hyperactivation motility characteristics; the amplitude of lateral head displacement > 7.5 microns, curvilinear velocity > 70 microns/s and linearity of < 30%. For this pilot study, 16 patients (eight in each group) were recruited. Using subzonal insemination (SUZI), up to five (mean = 4.4 +/- 0.3) spermatozoa selected using computer-image sperm selection (CISS) were micro-injected, or up to 15 (mean = 12.8 +/- 1.3 SD) unselected spermatozoa. In the group which utilized CISS, 28 out of 49 (57%) oocytes were fertilized compared with 13 out of 52 (25%) utilizing conventional SUZI (P < 0.04); polyspermy was 20% (n = 10) and 2% (n = 1) respectively. CISS with SUZI showed increased efficiency in achieving fertilization and is a novel approach to studying individual sperm function in a sperm egg bioassay where gamete ratios are close to unity.

Differential regulation of insulin-like growth factor binding protein-1 and -2 by insulin in the baboon (Papio anubis) endometrium

BackgroundThe purpose of this study was to examine the effect of insulin on expression and synthesis of IGFBP-1 and IGFBP-2 in the baboon endometrium in vitro.MethodsBaboon endometrial explants collected from cycling, ovariectomized, steroid-treated, simulated-pregnant and pregnant animals were cultured for 48 h in the presence or absence of insulin, with or without estradiol, progesterone and hCG.ResultsInsulin clearly inhibited IGFBP-1 production and mRNA expression in a time- and dose-dependent manner, whereas IGFBP-2 synthesis was not significantly affected. The inhibitory effects of insulin on IGFBP-1 were more evident in explants of non-pregnant tissue or tissue away from the implantation site. In the absence of insulin, synthesis of IGFBP-1 was induced in explants with low levels of de novo synthesis whereas IGFBP-2 synthesis was inhibited. This effect was potentiated by steroids and hCG in the explant cultures.ConclusionInsulin differentially regulates endometrial IGFBP-1 and IGFBP-2 secretion in the baboon.

Low Plasma Levels of hCG After 10,000-IU hCG Injection Do Not Reduce the Number or Maturation of Oocytes Recovered in Patients Undergoing Assisted Reproduction

Purpose:Our purpose was to determine whether there is a correlation between human chorionic gonadotropin (hCG) blood levels and oocyte maturation.Methods:Three samples of blood were obtained at different times from hCG administration as follows: 12 hr, 36 hr, during oocyte recovery, and at 84 hr, when the patient comes for embryo transfer.Results:A total of 5036 oocytes was retrieved from 404 patients prospectively recruited between April 1996 and March 1997. The percentage of metaphase-II oocytes at different blood levels ranged from 84 to 88%. The general trend does not show any significant increase in percentage of metaphase-II oocytes in association with an increasing serum hCG concentration.Conclusions:The results of this study suggest that at 12, 36, and 84 hr after hCG administration, levels as low as 50, 45, and 9 IU/L of hCG, respectively, are equally potent as higher levels at initiating maximal oocyte maturity.

Action of hypoxanthine and meiosis-activating sterol on oocyte maturation in the mouse is strain specific

Follicular fluid meiosis-activating sterol (FF-MAS) is regarded as an important compound relevant to meiotic resumption in mammalian oocytes. The objective of this study was to investigate the influence of FF-MAS on germinal vesicle breakdown (GVBD) and first polar body (PBI) extrusion with regard to culture conditions, state of the oocyte and mouse strain. Denuded oocytes (DO) and cumulus-enclosed oocytes (CEO) were retrieved from PMSG-primed Quackenbush or C57BL/6J x DBA/2 (C57) mice and cultured for 20 h in alpha-MEM medium under the following conditions: (i) 250 micromol/l dibutyryl cAMP (dbcAMP) +/- EGF, 1 ng/ml or FF-MAS, 20 micromol/l; (ii) 4 mmol/l hypoxanthine (HX) +/- EGF or FF-MAS; (iii) HX + EGF + FF-MAS; and (iv) HX + FF-MAS 5 h priming and subsequent culture with HX + EGF. Oocyte GVBD and PBI emission were recorded and stained with Hoechst 33342. Very limited meiotic inhibition was observed in Quackenbush mice in comparison with C57 mice. FF-MAS promoted maturation in C57 DO and CEO and Quackenbush DO. In Quackenbush DO and CEO and C57 DO a significant increase in atypical PBI extrusion occurred, but not in C57 CEO as well as in EGF-treated Quackenbush CEO primed or co-cultured with FF-MAS. These results support a meiosis resumption function for FF-MAS and suggest that in its presence, the quality of the MII oocytes retrieved appears to be influenced by the strain of the mice, the state of the oocyte and the presence or absence of growth factors in the culture medium.