Biochemical markers of placental dysfunction in assisted conception.

A possible mechanism for poor perinatal outcomes in singleton pregnancies conceived following assisted reproductive technologies (ART) and those conceived naturally following a period of infertility (>12 months) is thought to be placental dysfunction. This was investigated by measuring plasma concentrations of biochemical markers: (i) soluble fms-like tyrosine kinase1 (sFlt1); (ii) placental growth factor (PlGF); (iii) leptin; and (iv) plasminogen activator inhibitor 2 (PAI-2), serially at four antenatal time points. Baseline concentrations of each marker after delivery were also measured. The control group was naturally conceived singleton pregnancies with no history of infertility. Non-smoking, age-matched nulliparous women with no significant medical history were recruited to all groups. The ART group had significantly lower mean plasma concentrations of PlGF at all antenatal time points compared to the control group (p < 0.001). The subfertility (SF) group had significantly higher mean serum concentrations of leptin than the other groups at all time points (p < 0.001), even after correction for body mass index. There were no significant differences in sFlt1 and PAI-2 concentrations between the groups. Low plasma PlGF concentrations in the ART group might suggest abnormal placentation and/or abnormal function in ART pregnancies with relevance to pathogenesis of pregnancy complications in these women.

Fetal habituation in assisted conception.

BACKGROUND: Neurodevelopment outcomes of children conceived by Assisted Reproductive Technology (ART)have been the subject of much recent attention. To date there are no reports of neurodevelopmental performance before birth in this group. AIMS: To compare habituation (a measure of brain function) in fetuses conceived by assisted reproduction techniques (ART) with naturally conceived (NC) fetuses. STUDY DESIGN: Case control study. SUBJECTS: Women with singleton pregnancies matched for maternal age, parity and smoking were recruited in 2 groups: ART (n=20) and NC (n=20). OUTCOME MEASURES: Sound stimuli (250 Hz, 110 dB) at 10 second intervals lasting 2 s were administered to the fetus. The end point was habituation (cessation of movement for five consecutive stimuli) or a maximum of 30 stimuli. Responses of the fetus were observed with ultrasound at 28, 32 and 36 weeks' gestation, video-recorded and anonymised for analysis. RESULTS: At 28 weeks' gestation significantly more ART fetuses responded to sound of 250 Hz, 110 dB (p=0.02) but this difference did not persist at 32 and 36 weeks'. There was a significant increase in nonresponders as gestation advanced in the ART group. There was no difference in habituation or mean number of trials to habituate at all three gestations. CONCLUSIONS: ART fetuses demonstrated no differences in habituation suggesting that there is no neurodevelopment delay. However, a decrease in response to sound as gestation advances might be a harbinger for poor perinatal outcomes and needs exploration.

Spermatogenic and sperm quality differences in an experimental model of metabolic syndrome and hypogonadal hypogonadism.

The synergistic effect of the co-morbidities that comprise metabolic syndrome (MetS) is increasingly being recognised as an important contributor in the pathology of a broad spectrum of seemingly disparate conditions. However, in terms of male reproductive function, beyond erectile dysfunction, little is known about the influence of this cohort (collectively or separately) on spermatogenesis and sperm quality. The aims of this study were to assess the reproductive tract of a MetS animal model for detrimental changes, to determine whether a group of compounds (advanced glycation end products and their receptor) known to cause cell dysfunction and DNA damage was present and assess whether hypogonadotropic hypogonadism was the main contributing factor for the changes seen. Animals fed a high-fat diet were found to have significantly increased cholesterol, triglycerides, blood glucose, mean arterial pressure and visceral fat levels. Although serum testosterone was decreased, no changes were seen in either testicular or epididymal histology. Immunolocalisation of N(ε)-carboxymethyl-lysine and the receptor for advanced glycation end products was found in the testes, epididymides and sperm of the two treated groups of animals; however, ELISA did not show any difference in protein levels. Similarly, assessment of sperm nuclear DNA (nDNA) fragmentation by acridine orange test did not find significant differences in nDNA integrity. We conclude that the minimal effect on spermatogenesis and sperm quality seen in our model is probably due to the moderate increase of blood glucose rather than the hypogonadism.

Reduced sperm yield from testicular biopsies of vasectomized men is due to increased apoptosis.

OBJECTIVE: To compare sperm yields, apoptotic indices, and sperm DNA fragmentation from vasectomized men and fertile men undergoing vasectomy. DESIGN: Testicular biopsies from vasectomized (n = 26) and fertile men (n = 46), were milked to calculate sperm/gram and also formalin-fixed to determine the numbers of developing sperm and incidence and intensities of testicular FasL, Fas, Bax, and Bcl-2. Testicular sperm DNA fragmentation was assessed using the alkaline Comet assay. SETTING: An ART unit. PATIENT(S): Twenty-six men attending for intracytoplasmic sperm injection (ICSI) and 46 men attending for vasectomies. MAIN OUTCOME MEASURE(S): Spermatocyte, spermatid and sperm yields, Fas, FasL, and Bax staining. RESULT(S): Sperm yields from men vasectomized >5 years previously were markedly reduced compared to fertile men. Increased intensities of FasL and Bax staining were observed in the seminiferous tubules of vasectomy men. FasL positivity (percentage) also increased in Sertoli cells, and both FasL and Fas positivity (percentage) increased in primary spermatocytes and round spermatids of vasectomized men. Sperm DNA fragmentation, an end point marker of apoptosis, increased significantly in vasectomized men compared to fertile men. CONCLUSION(S): Reduced sperm yields after vasectomy are associated with increased apoptosis through the Fas-FasL and Bax pathways. Sperm after vasectomy displayed increased DNA fragmentation.

Soluble vascular endothelial growth factor receptor-1 (sFlt-1) is increased throughout gestation in patients who have preeclampsia develop.

OBJECTIVE: This study was undertaken to analyze prospectively circulating vascular endothelial growth factor (VEGF) and its soluble receptor, (s) Flt-1, throughout normotensive and preeclamptic pregnancies and to assess the importance of these proteins in the development of preeclampsia. STUDY DESIGN: In this longitudinal cohort study, serum samples were collected from recruited subjects throughout pregnancy at 12, 20, 30, and 37 weeks and in the 24 hours before and after delivery. Subjects were divided retrospectively into normotensive and preeclamptic groups. Circulating VEGF and sFlt-1 concentrations were analyzed by radioimmunoassay and enzyme-linked immunosorbent assay, respectively. RESULTS: Circulating sFlt-1 and VEGF significantly increased as gestation progressed and both were further elevated in preeclampsia compared with normotensive pregnancy. Soluble Flt-1 concentrations were elevated early in gestation and were significantly increased at 30 weeks' gestation in those who subsequently developed preeclampsia. CONCLUSION: These results indicate a definite association between elevated sFlt-1 concentrations and the onset of preeclampsia suggesting that sFlt-1 is linked with disease pathogenesis.

Incidence of Fas positivity and deoxyribonucleic acid double-stranded breaks in human ejaculated sperm.

OBJECTIVE: To determine the incidence of Fas positivity and DNA double-strand breaks (DSB) as indicators of early- and late-stage apoptosis in ejaculated sperm. DESIGN: Fas positivity was assessed by flow cytometry and DSB by the neutral Comet assay. SETTING: Andrology Laboratory, Royal Maternity Hospital, Belfast, Northern Ireland, United Kingdom. PATIENT(S) AND INTERVENTION(S): Forty-five infertile men undergoing infertility investigations and 10 fertile men undergoing vasectomies. MAIN OUTCOME MEASURE(S): Percentage Fas-positive cells, percentage DNA fragmentation, olive tail moment. RESULT(S): The apoptotic marker Fas was detected in ejaculated sperm, with a higher incidence of Fas positivity in teratozoospermic and asthenozoospermic than in normozospermic semen. No Fas positivity was observed in fertile men's sperm. Deoxyribonucleic acid fragmentation (DSB) was greater in infertile than in fertile men's sperm and also greater in sperm in semen than in sperm prepared for assisted conception. There was an inverse relationship between DSB and both sperm concentration and motility. There was no relationship between Fas positivity and DNA damage. CONCLUSION(S): Fas was expressed in sperm of infertile men. In contrast, DNA fragmentation was observed in all sperm of fertile and infertile men and correlated with inadequate concentration and motility, which suggests that sperm DSB are ubiquitous and are not solely associated with apoptosis.

Effects of cryopreservation on testicular sperm nuclear DNA fragmentation and its relationship with assisted conception outcome following ICSI with testicular spermatozoa.

The objective of the study was to investigate the effects of freeze-thawing on testicular sperm DNA fragmentation, fertilization rates and pregnancy rates following intracytoplasmic sperm injection with testicular spermatozoa (TESE). This ongoing prospective study included 88 couples attending for infertility treatment where the man presented with obstructive azoospermia at the Regional Fertility Centre, Belfast, UK. Patients were allocated to receive TESE treatment with fresh or freeze-thawed spermatozoa. Sperm aliquots were stored in liquid nitrogen at -196 degrees C following static phase vapour cooling or cooling at controlled rates using a programmable freezer. Samples were thawed at either room temperature or 37 degrees C. Sperm nuclear DNA; assessed by the alkaline Comet assay, was significantly damaged by slow freezing followed by fast thawing. Pregnancies were more likely to be achieved with spermatozoa displaying markedly less DNA damage. However, no differences were observed in the fertilization rates, the number of blastomeres or the cumulative embryo score between TESE cycles using either fresh or frozen thawed testicular spermatozoa. The pregnancy rates tended to be higher following fresh TESE cycles (30%) compared with TESE cycles using frozen-thawed testicular spermatozoa (26%), although this difference did not reach statistical significance. It is concluded that cryopreservation of testicular spermatozoa may reduce pregnancy rates, although this will only be confirmed by a much larger multi-centre trial.

The hidden impact of diabetes on male sexual dysfunction and fertility.

Diabetes affects an increasingly large number of young men of reproductive age. Erectile and ejaculatory difficulties arise due to vascular and neuropathic problems. The treatment of these may have effects on fertility potential. Erectile dysfunction can be treated with mechanical devices and intracavernosal injections. Although these have not been shown to affect fertility directly, they may result in poor compliance and hence reduced frequency of ejaculation with subsequent deterioration in sperm quality. Other medical treatments may have a more direct effect. The phosphodiesterase (PDE) inhibitor pentoxifylline has been shown to affect sperm quality and early embryo development. Therefore, Viagra, also a PDE inhibitor, may affect sperm quality. There is conflicting evidence about this in the literature. Ejaculatory difficulties are also more common in diabetics although treatments such as Trucut testicular biopsy and intracytoplasmic sperm injection have improved the outlook for these patients. There is also some evidence that spermatogenesis is affected by diabetes and that patients have a reduced sperm motility and semen volume. Therefore, diabetes has a significant impact on the fertility of men with this disease both directly and indirectly. The extent of iatrogenic influence on the reduced fertility potential of these patients needs to be researched as a matter of urgency.

Chromogranin A proteolysis to generate beta-granin and WE-14 in the adenohypophysis during the rat oestrous cycle.

Immunohistochemical analysis of the male and female rat adenohypophysis revealed that chromogranin A (CgA), beta-granin and WE-14 immunostaining was localised to follicle stimulating hormone (FSH) producing cells, while luteinizing hormone (LH) producing cells exhibited chromogranin A and beta-granin immunostaining. The intensity of chromogranin A, beta-granin and WE-14 immunostaining exhibited variation during the oestrous cycle; weak immunostaining was observed during proestrous and oestrous, corresponding with the lowest cellular concentration of luteinizing and follicle stimulating hormone. Chromogranin A and beta-granin immunostaining were similar in both the male and female (at dioestrous), however, a larger number of more intense WE-14 immunopositive cells were evident in the male adenohypophysis relative to the female at any stage of the cycle. The tissue and plasma concentrations of beta-granin and WE-14 immunoreactivity fluctuated throughout the oestrous cycle. Maximum and minimum beta-granin and WE-14 tissue concentration counterpoised the latent maximum and minimum plasma concentration. Chromatographic analysis of adenohypophysis extracts revealed the degree of chromogranin A proteolysis throughout the oestrous cycle; in contrast, plasma profiles consistently possessed a large chromogranin A-like immunoreactant. This data suggests that chromogranin A biosynthesis, proteolysis and the secretion of its derived peptides parallels that of the gonadotroph hormones throughout the oestrous cycle.

Preventing ovarian hyperstimulation syndrome by inhibiting the effects of vascular endothelial growth factor.

OBJECTIVE: To evaluate whether inhibiting the effects of vascular endothelial growth factor can prevent the ovarian hyperstimulation syndrome. STUDY DESIGN: Rates were hyperstimulated with follicle-stimulating hormone injections. On the final day of stimulation, the rats were randomized to receive or not receive exogenous soluble fmslike tyrosine kinase (sFlt-1). Forty-eight hours later capillary permeability was determined by measuring the concentration of Evans blue dye (EB) in peritoneal irrigation fluid 3 minutes after an intravenous injection of EB. RESULTS: The peritoneal EB level was statistically significantly lower in the hyperstimulated group, which received the sFlt-1, than in the stimulation-only group. CONCLUSION: sFlt-1 may play a therapeutic role in management of the ovarian hyperstimulation syndrome by specifically inhibiting the effects of vascular endothelial growth factor.

Variations in serum vascular endothelial growth factor binding profiles and the development of ovarian hyperstimulation syndrome.

OBJECTIVE: To compare the ability of serum to sequester vascular endothelial growth factor (VEGF) among patients who did and did not develop ovarian hyperstimulation syndrome (OHSS). DESIGN: Prospective, observational study. SETTING: A regional fertility centre with a commitment to research. PATIENT(S): Five patients undergoing controlled ovarian hyperstimulation as part of an in vitro fertilization cycle who developed severe OHSS, and five controls. INTERVENTION(S): Serum, collected at the time of oocyte retrieval, was incubated with radioactive VEGF (125I-VEGF(165)) for 2 hours before being passed down a sephadex G-150 gel filtration column. The fractional radioactive profile was then determined. MAIN OUTCOME MEASURE(S): The distribution of radioactive VEGF across the various fractions was measured in serum samples obtained from the two groups. RESULT(S): The 125I-VEGF(165) applied to the column eluted in two peaks centered on fractions 18 +/- 2 and 39 +/- 2. The molecular weight in the first peak was >300,000 kDa and represented "bound" VEGF; the second peak represented "unbound" VEGF. In the OHSS group, there was statistically significantly less radioactivity in the first peak than in the no-OHSS group. CONCLUSION(S): Patients who do not develop OHSS appear to have a high-molecular-weight protein that binds VEGF to a greater degree than occurs in patients who develop OHSS.

Ovarian hyperstimulation syndrome and assisted reproductive technologies: why some and not others?

BACKGROUND: alpha(2)-Macroglobulin (alpha(2)M) is a multifactorial binding protein, found in follicular fluid, that is a naturally occurring inhibitor of vascular endothelial growth factor (VEGF). The aim of this study was to determine if there is a relationship between serum VEGF levels, alpha(2)M levels and the development of OHSS in hyperstimulated subjects undergoing IVF (those with 15 or more oocytes). METHODS: Venous blood was collected at the time of oocyte retrieval from subjects who yielded 15 or more oocytes. Serum samples were analysed for VEGF and alpha(2)M concentrations. RESULTS: There was no statistically significant difference in serum VEGF levels at the time of oocyte retrieval between hyperstimulated subjects who did and did not subsequently develop OHSS [3.95 (3.3-4.4) versus 3.85 (3.3-4.5); P = 0.79]. By contrast, the serum level of alpha(2)M was statistically significantly higher in the group of subjects who did not develop OHSS [2.27 (1.91-2.58) versus 1.67 (1.45-1.73)]. CONCLUSIONS: These results suggest that elevated alpha(2)M levels are associated with a decreased risk of developing OHSS. alpha(2)M may act by 'removing and inactivating' VEGF, with higher levels providing increased protection against the syndrome. alpha(2)M measurements may help to differentiate those for whom it is safe to proceed with embryo transfer from those for whom it is not, because of the risk of OHSS.