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.

Is assisted reproduction associated with abnormal placentation?

Artificial reproductive technologies (ART) and conception following a period of untreated infertility (>1 year) are independently associated with increased pregnancy complications in both singleton and multiple pregnancies. It is unknown if placental dysfunction associated with macroscopic and/or microscopic histological discrepancies might explain some of these variances. Our aim was to compare the histopathology of placentae from singleton pregnancies belonging to 3 groups, as follows: conception as a result of ART; spontaneous conception (<1 year of trying); and conception following untreated infertility (>1 year). Pathological examination of placentae from singleton pregnancies of nonsmoking, age-matched primiparous women with no significant medical history and no known uterine congenital anomalies was performed by a single pathologist blinded to the groups. Features were compared using analysis of variance and chi-square tests. A total of 89 placental pathology reports were available (control  =  39, infertility  =  17, ART  =  33). The mean placental thickness was significantly higher in the ART group when compared to the spontaneous conception group (P  =  0.02). There were significantly more placental hematomas in the ART group (P  =  0.04) compared to the other groups. There were no significant differences in rates of abnormal placental shapes or abnormal cord insertions. There were no statistically significant differences in the incidence of microscopic placental lesions, nor were there any statistically significant differences in the incidence of macroscopic and microscopic placental lesions between the infertility group and the other groups. Placentae of ART pregnancies show significantly increased thickness and a higher incidence of hematomas. Increased placental thickness has previously been linked to increased perinatal risk.

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.

Male diabetes mellitus and assisted reproduction treatment outcome.

The long-held view that diabetes has little effect on male reproductive function has been challenged by findings that the condition influences fertility in numerous previously undetected ways. This retrospective chart review of 3000 couples determined the incidence of couples with a male diabetic seeking assisted reproduction treatment and assessed any relationship between male diabetes and IVF/intracytoplasmic sperm injection (ICSI) outcome. Eight (2.7%) couples were found with a diabetic male partner, of which 18 couples underwent assisted reproduction treatment (five IVF, 12 ICSI, one both), with fertilization rates (IVF 68%, ICSI 62%) similar to non-diabetic patients (IVF 70%, ICSI 71%) and no difference in embryo quality. Two men had retrograde ejaculation and two were azoospermic. Other than reduced sperm motility, the remaining 14 had normal World Health Organization semen parameters. Embryo transfers produced one pregnancy (5% combined IVF/ICSI pregnancy rate/cycle) giving a lower-than-expected rate (28.8%). The pregnancy rate from seven FETs (29%) was comparable to the expected (21.3%). Compared with non-diabetics, approximately three times more couples with diabetic men sought treatment, with a larger percentage having 'unexplained' infertility. Fertilization rates and embryo quality did not differ but pregnancy rates were lower in couples with a diabetic male.

The influence of type 1 diabetes mellitus on spermatogenic gene expression.

Routine semen analysis found no differences in diabetic men; however, mRNA profiles showed changes in the expression of genes involved in oxidative stress.

Sildenafil citrate (Viagra) impairs fertilization and early embryo development in mice.

OBJECTIVE: To determine the effects of sildenafil citrate, a cyclic monophosphate-specific type 5 phosphodiesterase inhibitor known to affect sperm function, on fertilization and early embryo cleavage. DESIGN: This acute mammal study included male and female mice assigned randomly, the females sacrificed after mating and their oocytes/embryos evaluated at four time periods after treatment. SETTING: Academic research environment. ANIMAL(S): Male and female CBAB(6) mice. INTERVENTION(S): Female mice were injected intraperitoneally with 5 IU gonadotropin (hCG) to stimulate follicular growth and induce ovulation. They were each caged with a male that had been gavaged with sildenafil citrate (0.06 mg/0.05 mL) and allowed to mate. After 12, 36, 60, and 84 h, females were killed, their oviducts were dissected out, and retrieved embryos were assessed for blastomere number and quality. MAIN OUTCOME MEASURE(S): Fertilization rates and numbers of embryos were evaluated after treatment. RESULT(S): Fertilization rates (day 1) were markedly reduced (-33%) in matings where the male had taken sildenafil citrate. Over days 2-4, the numbers of embryos developing in the treated group were significantly fewer than in the control group. There was also a trend for impaired cleavage rates within those embryos, although this did not reach significance. CONCLUSION(S): The impairments to fertility caused by sildenafil citrate have important implications for infertility centers and for couples who are using this drug precoitally while attempting to conceive.

Are pregnancy rates compromised following embryo freezing to prevent OHSS?

OBJECTIVE: To compare pregnancy rates with fresh and frozen embryo transfer in patients admitted to Royal Jubilee Maternity Service (RJMS), Belfast between January 1st 2004 and December 31st 2005 with ovarian hyperstimulation syndrome (OHSS). METHOD: A retrospective analysis of all ART cycles (2,283) carried out in RJMS between January 1st 2004 and December 31st 2005 and of all patients admitted to RJMS within 3 weeks of assisted reproduction therapy (ART). RESULTS: The incidence of OHSS requiring admission was 2.01%, which represented 80.70% of post-ART emergency admissions. The eventual pregnancy rate was 52.27% in all women admitted with OHSS. The pregnancy outcome in OHSS patients who received fresh embryo transfer was 56.52% and with frozen embryo transfer 50%. The main indications for fertility treatment in OHSS cases were male factor (31%) and polycystic ovarian syndrome (14%). Two distinct incidence peaks of OHSS were identified--early and late. 77.77% of women who suffered from late onset OHSS had a concurrent positive pregnancy test. CONCLUSION: The pregnancy rate in OHSS cases, both with fresh and subsequently with frozen embryo transfer, was exceptionally high. There was no statistically significant difference between fresh and frozen embryo transfer pregnancy rates. An elective embryo freezing policy to moderate the severity and duration of OHSS does not compromise outcome for women at risk of OHSS.

Distribution of the receptor for advanced glycation end products in the human male reproductive tract: prevalence in men with diabetes mellitus.

BACKGROUND: Diabetics have a significantly higher percentage of sperm with nuclear DNA (nDNA) fragmentation and increased levels of advanced glycation end products (AGEs), in their testis, epididymis and sperm. As the receptor for AGEs (RAGE) is important to oxidative stress and cell dysfunction, we hypothesise, that it may be involved in sperm nDNA damage. METHODS: Immunohistochemistry was performed to determine the presence of RAGE in the human testis and epididymis. A comparison of the receptor's incidence and localization on sperm from 10 diabetic and 11 non-diabetic men was conducted by blind semi-quantitative assessment of the immunostaining. Enzyme-linked immunosorbent assay analysis ascertained RAGE levels in seminal plasma and sperm from 21 diabetic and 31 non-diabetic subjects. Dual labelling immunolocalization was employed to evaluate RAGE's precise location on the sperm head. RESULTS: RAGE was found throughout the testis, caput epididymis, particularly the principle cells apical region, and on sperm acrosomes. The number of sperm displaying RAGE and the overall protein amount found in sperm and seminal plasma were significantly higher in samples from diabetic men (P < 0.01, P < 0.0001 and P < 0.0001, respectively). CONCLUSIONS: The presence of RAGE implies that it may play a central role in sperm nDNA damage particularly in diabetic men where the levels are elevated.

Sildenafil citrate improves sperm motility but causes a premature acrosome reaction in vitro.

OBJECTIVE: To determine whether sildenafil citrate, a cyclic monophosphate-specific type 5 phosphodiesterase inhibitor, influences sperm motility or the acrosome reaction. DESIGN: Laboratory analysis of sperm motility after exposure to sildenafil citrate using computer-assisted semen analysis and acrosome reaction by fluorescein isothiocyanate-labeled peanut agglutinin staining. SETTING: An assisted reproductive technology (ART) unit. PATIENT(S): Fifty-seven male patients. INTERVENTION(S): Sperm were divided into 90% (those with the best fertilizing potential used in assisted conception) and 45% (the poorer population) fractions by density centrifugation and incubated with sildenafil citrate (0.67 muM) at 37 degrees C for up to 180 minutes. MAIN OUTCOME MEASURE(S): Both the number and velocity of progressively motile sperm were significantly increased by sildenafil citrate between 15 and 135 minutes. Furthermore, samples revealed that these effects were consistent in the 90% and 45% populations of sperm. In both populations, sildenafil also caused a significant increase in the proportion of acrosome-reacted sperm-22.1% compared with 11.8% in the control group of the good quality fraction and 16.6% compared with 9.4% in the control group of the poorer quality fraction. CONCLUSION(S): The use of sildenafil citrate may adversely affect male fertility.

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.

Effects of delta-9-tetrahydrocannabinol, the primary psychoactive cannabinoid in marijuana, on human sperm function in vitro.

OBJECTIVE: To investigate effects of delta-9-tetrahydrocannabinol (THC) on human sperm function in vitro. DESIGN: Laboratory analysis of sperm motility after exposure to THC using computer-assisted semen analysis and acrosome reaction by fluoroscein isothiocyanate-labeled peanut agglutinin staining. SETTING: An assisted reproductive technology unit. PATIENT(S): Seventy-eight male patients. INTERVENTION(S): Sperm were divided into 90% (the best fertilizing potential used in assisted conception) and 45% (the poorer subpopulation) fractions by density centrifugation and incubated with THC at concentrations equivalent to therapeutic (0.032 microM) and recreational (0.32 and 4.8 microM) plasma levels at 37 degrees C for 3 h. MAIN OUTCOME MEASURE(S): Sperm motility and spontaneous and induced acrosome reactions. RESULT(S): Percentage progressive motility was decreased dose dependently in the 90% fraction (by 2%-21%; P<.05; P<.001). The 45% fraction showed a greater decrease in percentage progressive motility (by 28% at 0.032 microM; 56% at 4.8 microM; P=.004 and P=.01 res). Straight line velocity and the average path velocity also were reduced (by 10%, in the 90% LAYER) in both fractions. Spontaneous acrosome reactions were reduced in the 90% (17% at 0.032 microM, 35% at 4.8 microM P=.004 and P<.001 resp) and more markedly in the 45% fractions (17%-35%; P<.001). When the acrosome reaction was artificially induced (90% fraction) by A23187, THC (4.8 microM) resulted in a 57% inhibition (P<.001). CONCLUSION(S): The use of THC as a recreational drug may adversely affect male fertility.

Dietary oestrogens and male fertility potential.

Reports of increased incidences of male reproductive abnormalities and falling sperm counts have prompted interest into the nature of these threats to global fertility. Xenoestrogens have been flagged as major culprits but to date, little is known about the effects of dietary phytoestrogens on male reproductive health. These non-steroidal oestrogens of plant origin are potent endocrine disruptors that modulate normal physiological functions. Phytoestrogens have become a major component in the typical Western fast food diet over the last few decades. Soy formula milk is another common source of phytoestrogens, now used increasingly as an alternative to breast or cow's milk for infants with allergies. This use is of particular concern since the most vulnerable periods for oestrogenic insult are thought to be the pre- and neonatal periods when irreversible damage can be inflicted on the developing germinal epithelium. Studies into the safety of phytoestrogens are urgently needed either to allay fears or increase awareness of the effects of our modern diet on future fertility.

Effects of short and long incubations on DNA fragmentation of testicular sperm.

DNA fragmentation in testicular sperm from men with obstructive azoospermia is increased by 4-hour and 24-hour incubations and after cryopreservation with the effect is intensified by post-thaw incubation. Testicular sperm for use in intracytoplasmic sperm injection (ICSI) should be injected without delay.

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.

Vulvodynia.

CONTEXT: Vulvodynia is a term used to describe chronic burning and/or pain in the vulva without objective physical findings to explain the symptoms. The terminology and classification of vulvodynia continue to evolve, and much remains to be understood about the prevalence, pathogenesis, natural history, and management of this distressing condition. STARTING POINT: James Aikens and colleagues showed that chronic vulval pain (vulvodynia or vulvar dysaesthesia) is associated with worse depressive symptoms (Am J Obstet Gynecol 2003; 189: 462-66). However, the increased scores for depression in this case-control study were attributed to sexual disinterest and experience of chronic pain rather than to features of depressive disorder. These results lend weight to the increasing need for better understanding of the pathogenesis of vulval pain and how to manage it appropriately. WHERE NEXT? The aetiology of vulvodynia and effectiveness of treatments need further study. Appraising the available literature, we have formulated a useful approach to patients with chronic vulval pain. There is a pressing need for further case-control studies of potential causes of vulvodynia and for randomised trials of interventions.

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.