The World Health Organization developed a time to pregnancy (TTP) study (number of menstrual cycles taken to conceive) to determine whether the average TTP is increasing and semen quality decreasing with time. The present study describes clinical, semen and hormone characteristics obtained from male partners of pregnant women in Melbourne, Australia, and examines the associations between these characteristics.
Male partners (n = 225) of pregnant women (16–32 weeks) who conceived naturally had physical examination, health and lifestyle questionnaires, semen and hormone (FSH, LH, sex hormone-binding globulin, testosterone and Inhibin B) analyses.
Previously known associations between semen, hormone and clinical variables were confirmed as significant: sperm numbers (concentration and total sperm count) correlated positively with Inhibin B and inversely with FSH and left varicocele, while total testicular volume correlated positively with sperm numbers and Inhibin B and inversely with FSH. However, only abstinence, total testicular volume, varicocele grade and obesity (BMI > 30 kg/m2) were independently significantly related to total sperm count. Compared with those with BMI < 30 (n = 188), obese subjects (n = 35) had significantly lower total sperm count (mean 324 versus 231 million, P = 0.013) and Inhibin B (187 versus 140 pg/ml, P < 0.001) but not FSH (3.4 versus 4.0 IU/l, P = 0.6).
Obese fertile men appear to have reduced testicular function. Whether this is cause or effect, i.e. adiposity impairing spermatogenesis or reduced testicular function promoting fat deposition, remains to be determined.
We have previously suggested that the Toluidine Blue (TB) test can be used for sperm chromatin structure assessment. In this study, we wished to evaluate the clinical applicability of the TB test in assessing male fertility potential using well-defined groups of fertile and infertile men.
Sixty-three fertile and 79 infertile men were tested. Infertility thresholds for the proportion of sperm with abnormal [TB dark cells (TBDCs)] and normal [TB light cells (TBLCs)] chromatin structure were set by the ROC curve analysis.
Thresholds of 45% TBDC and 20% TBLC were highly predictive for infertility (specificity of the test: 92 and 90%, respectively), but they were poor predictors of the fertility (sensitivity of the test: 42 and 32%, respectively). Odds ratio for infertility was 7.5 [95% confidence interval (CI): 2.7–20.8] when the 45% TBDC threshold was used and 4.4 (95% CI: 1.7–11.6) when the 20% TBLC threshold was used.
The TB test can be suggested for clinical use as a complementary test for standard semen analysis to diagnose male infertility.
Hepatitis B virus (HBV) has been determined to exist in semen and male germ cells from patients with chronic HBV infection, but no data are yet available on the impact of HBV S protein (HBs), the main component of HBV envelop protein, on the human reproductive system. The purpose of this article was to investigate the effect of HBs on human sperm function.
Sperm motility analyses, sperm penetration assays, mitochondrial membrane potential assays, immunolocalizations with confocal microscopy and flow cytometry analyses were performed.
HBs reduced sperm motility in a dose- and time-dependent manner and caused the loss of sperm mitochondrial membrane potential. HBs–HBs monoclonal antibody (MAb) complex apparently aggravated such impairments. After 4 h incubation with HBs at concentrations of 25, 50, 100 µg/ml, the percentages of sperm motility a+b significantly decreased compared with the control (P < 0.01). The fertilization rate and the fertilizing index in HBs-treated group were 40% and 0.57, respectively, which were significantly lower than 90% and 1.6, respectively, in the control (P < 0.01). The asialoglycoprotein receptor (ASGP-R) and HBs were found to localize mainly on the postacrosomal region. Both ASGP-R MAb and asialofoetuin, a high-affinity ligand of ASGP-R, inhibited the HBs-caused loss of sperm motility and mitochondrial membrane potential.
HBs had adverse effects on human sperm function, and ASGP-R may play a role in the uptake of HBs into sperm cells, as demonstrated by the competitive inhibition of ASGP-R MAb or asialofoetuin, resulting in diminished impairment caused by HBs.
Unilateral orchidectomy in monkeys increases spermatogenesis in the remaining testis in association with elevated follicle-stimulating hormone (FSH) secretion and testicular testosterone. The present study examined the relative importance of FSH and testosterone in driving the primate testis toward its spermatogenic ceiling.
Adult male rhesus monkeys were treated with a gonadotropin-releasing hormone receptor antagonist to inhibit endogenous FSH and luteinizing hormone (LH) secretion. The gonadotrophin drive to the testis was replaced with a pulsatile recombinant human FSH and LH infusion to maintain testicular volume and circulating testosterone and inhibin B at physiological levels. A selective monotropic elevation of FSH or LH that doubled the concentrations of inhibin B or testosterone, respectively, was then imposed for 4 weeks, each in a group of four monkeys. In a third group (n = 4), the gonadotrophin drive remained clamped at physiological levels. Bromo-deoxyuridine was administered 3 h prior to castration, and the effects of the monotropic hormone increments on germ cell number, S-phase labeling and degeneration were determined.
Increased FSH, but not LH, produced increases in testicular volume (P < 0.05), the proportion of A pale spermatogonia entering the cell cycle and the numbers of differentiated spermatogonia and more advanced germ cells (P < 0.05). Indexes for spermatogonia labeling and germ cell degeneration were not affected.
Under physiological conditions, circulating concentrations of FSH directly dictate sperm output of the primate testis by regulating the proportion of Ap spermatogonia in the growth fraction. An effect of FSH on survival of the first generation of differentiated B spermatogonia is not excluded.
Companion studies using an experimental non-human primate paradigm known as a testicular clamp indicated that the behavior of undifferentiated type A spermatogonia did not conform fully to earlier classical models. This issue was therefore re-examined in normal monkeys.
Adult male rhesus monkeys (n = 4) received an i.v. bolus of 5-bromo-2'-deoxyuridine (BrdU): one testis (first) was removed 3 h later and the remaining testis (second) was removed after 11 days and 3 h. Tissue was fixed in Bouin’s solution, and numbers of A dark (Ad), small A pale (Aps) and large A pale spermatogonia, differentiating B spermatogonia, S-phase-labeled and degenerating cells were enumerated. Data are given as mean ± SEM.
During the early stages of the seminiferous epithelial cycle in the first testis, Ap spermatogonia (1.3 cells/cross section) were predominantly Aps (nuclear dia., 7.1 ± 0.1 µm). Aps were never S-phase labeled. Apl (nuclear dia., 8.8 ± 0.5 µm) appeared in Stages IV–VI and were maximal in Stages VII–X when S-phase labeling of this phenotype at 3 h was greatest. The first generation of B spermatogonia appeared in Stages XI–XII (0.84 cells/cross section). Using cells/cross section, the ratio of Ap (Stages I–V):B1:B2:B3:B4:preleptotene spermatocyte was 1:0.7:1.4:2.8:5.6:11.2. In the second testis, labeled Aps (and Apl) were observed. Ad were not BrdU labeled, and degenerating cells were rarely observed.
The results are not entirely consistent with earlier models of spermatogonial proliferation and differentiation in the monkey. Most notably, our findings suggest that in any one cycle of the seminiferous epithelium only a fraction of Ap spermatogonia is mitotically active.
Levonorgestrel (LNG), as a dedicated emergency contraception (EC) product, has been available over-the-counter in China for 10 years. Until now, only a small number of deliveries after LNG–EC failure have been documented.
This study was a prospective comparative cohort study. A group of 332 pregnant women who had used LNG–EC during the conception cycle was recruited, and matched to a group of 332 pregnant women without the exposure to LNG. Congenital malformations, perinatal complications and delivery circumstances were investigated in this study.
There were 31 pregnant women in the study group and 28 in the comparison group miscarried within 14 weeks of gestation. In the study and comparison groups, four malformations were found in each group. In the study group, both birthweight (3416 versus 3345 g, P = 0.040) and the sex ratio of birth (boys/girls, 1.14 versus 0.90, P = 0.153) were higher than in the comparison group. There were no statistically significant differences in the incidence of miscarriage or malformation or in the neonatal outcome between the two groups.
There was no association between the use of LNG–EC pills and the risk of major congenital malformations, pregnancy complications or any other adverse pregnancy outcomes in our study.
The increasing use of emergency contraceptive pills (ECPs) does not seem to reduce the number of induced abortions as would be expected, indicating that women use ECPs might also be a strong factor affecting their final efficacy. The study aimed to understand the attitude towards, and use of, ECPs among women seeking an abortion.
A cohort study was conducted via face-to-face questionnaire interview among women seeking abortion in Shanghai, China. Logistic regression analysis and 2 test were performed for statistical analysis.
The response rate was 99.3%. Among all 5677 respondents aged 15–48 years, 48.8% were ECP ever-users. Compared with ever-users, ECP never-users were less likely to have used contraception during the present cycle of conception (P < 0.001). In response to the question on the main reason for non-use of contraception, ECP never-users were less likely to realize the risk of pregnancy and had less contraceptive knowledge (P < 0.001). Among 2773 ECP ever-users, 72.7% did not use ECPs to prevent the current pregnancy, mainly due to lack of awareness of pregnancy risk. Out of 757 women, 437 (57.7%) repeated unprotected sex after taking ECPs during the current pregnant cycle. A pharmacy was the preferred source to access ECPs, for the reason of convenience.
Non-use of ECPs was correlated to less knowledge on fertility and a lower rate of contraceptive use among abortion-seeking women. Women of reproductive age should have access to ECPs and receive sufficient information on their use. Health care providers and pharmacists should also be trained in contraceptive counselling, including ECPs.
The purpose of the study was to determine the influence of bowel endometriosis on fertility, and to study whether its removal improves fecundity in women with endometriosis-associated infertility.
Three groups of infertile patients were included in the study. Group A (60 women) consisted of patients who underwent surgery for endometriosis with colorectal segmental resection. In group B, 40 patients with evidence of bowel endometriosis underwent endometriosis removal without bowel resection. Group C consisted of 55 women who underwent surgery for moderate or severe endometriosis with at least one endometrioma and deep infiltrating endometriosis but without bowel involvement. The women were clinically evaluated before laparoscopy and then at 1 month, at 6 months and at each year up to 4 years after surgery. Main outcome measures were surgical complications as well as post-operative pregnancy rate, time to conception and monthly fecundity rate.
The monthly fecundity rates (MFR) in groups A, B and C were 2.3, 0.84 and 3.95%, respectively. The difference in the MFR between groups was significant (P < 0.05).
The presence of bowel infiltration by endometriosis seems to negatively influence the reproductive outcome in women with endometriosis-associated infertility. The complete removal of endometriosis with bowel segmental resection seems to offer better results in terms of post-operative fertility.
There seems to be little discussion between patient and physician about the use of complementary and alternative medicines (CAMs), perhaps because they are not perceived to have adverse effects on fertility. We therefore compared ongoing pregnancy and live birth rate in spontaneous users and non-users of CAM during a 12-month period of assisted reproduction technique (ART) treatment. Consent was by questionnaire return.
This was a prospective observational cohort study with a 12-month follow-up period, sampling consecutive patients attending five ART clinics in Denmark. N = 728 women about to have ART for the first time completed self-report assessments prior to treatment (Time 1, T1) and at 12-month follow-up (Time 2, T2). Data from treatment records were also available for n = 590.
About 30.6% (n = 223) of women used CAMs during the observation period. At T2 the ongoing pregnancy and live birth rate was 31.3% lower in CAM users (42.2%) compared with non-users (61.4%). Adjusted odds of pregnancy/live birth remained lower in CAM users versus non-users, odds ratio = 0.467 (95% confidence interval 0.306–0.711) after controlling for prognostic indicators (age, parity, years infertile).
Concurrent use of CAM during treatment with ART was associated with a 30% lower pregnancy rate that could not be explained by poor prognosis or life style factors. The mechanisms that could account for this association were discussed. Concurrent CAM use should be monitored during ART. A main limitation was that we could not ascertain which type of CAM was most associated with lower pregnancy rates.
Although elective single embryo transfer (eSET) minimizes the multiple birth rate after in vitro fertilization (IVF)/intra cytoplasmic sperm injection (ICSI), there remain concerns in many countries that it is less effective and more expensive than conventional double embryo transfer (DET).
We compared the clinical outcome achieved in the years 1995–1999, in which eSET was rarely used (4.2% of women, DET period) with that of the years 2000–2004, in which eSET was more widely used (46.2%, eSET period). In the DET period, 826 women had 1359 fresh embryo cycles followed by 589 frozen–thawed embryo transfer (FET) cycles. In the eSET period, 684 women had 1027 fresh and 683 FET cycles. The cumulative term live birth rate/woman was the primary clinical outcome measure. An incremental cost-effectiveness ratio of a term live birth was also calculated based on hospital charges and medication prices of IVF/ICSI treatment.
The cumulative pregnancy rate/oocytes pickup (38.2 versus 33.1%, P = 0.01), cumulative live birth rate/oocytes pickup (28.0 versus 22.5%, P = 0.002) and cumulative live birth rate/woman (41.7 versus 36.6%, P = 0.04) were all higher in the eSET period than in the DET period. The cumulative multiple birth rate was significantly lower in the eSET period than in the DET period (8.9 versus 19.6%, P < 0.0001). A term live birth in the eSET period was 19 889 euros less expensive than in the DET period.
This study shows that eSET with cryopreservation is more effective and less expensive than DET and should be adopted as a treatment of choice.
The pathology underlying recurrent implantation failures (RIF) is not clear and treatment options proposed are generally not evidence based. Although the effect of heparin on trophoblast biology has not been studied extensively, given the available data suggesting a possible beneficial effect of heparin on embryo implantation, we decided to undertake this pilot study.
One hundred and fifty women with ≥2 failed assisted reproduction treatment cycles were included in this randomized open-label pilot trial. Participants underwent controlled ovarian stimulation with the long protocol and were randomly allocated to receive 1 mg/kg/day low molecular weight heparin (LMWH) or no treatment in addition to routine luteal phase support (LPS) on the day after oocyte retrieval. LPS and LMWH was continued up to the 12th gestational week in pregnant participants.
There were 26 (34.7%) live births in the LMWH group, and 20 (26.7%) in the control group (absolute difference 8.0%, 95% CI –4.2 to 24.9%, P = 0.29). There were 34 (45.3%) and 29 (38.7%) clinical pregnancies in the LMWH and control groups, respectively (absolute difference 6.6%, 95% CI –9.0 to 21.8%, P = 0.41). Implantation rates were 24.5 and 19.8% in the LMWH and control groups, respectively (absolute difference 4.7%, 95% CI –4.7 to 14.1%, P = 0.33).
Despite lack of statistical significance, observed relative increase by 30% in live birth rates with LMWH may be regarded as a clinically significant trend necessitating further research on the use of empirical LMWH in women with RIF and possibly in all women undergoing assisted reproduction treatment. Failure to demonstrate statistical significance of the observed treatment difference may be due to limited sample size of this pilot study.
Clinicaltrials.gov registration number: nCT00750451.
A selective switch to elective single embryo transfer (eSET) in IVF has been suggested to prevent complications of fertility treatment for both mother and infants. We compared seven IVF strategies concerning their cost-effectiveness using a Markov model.
The model was based on a three IVF-attempts time horizon and a societal perspective using real world strategies and data, comparing seven IVF strategies, concerning costs, live births and incremental cost-effectiveness ratios (ICERs).
In order to increase pregnancy probability, one cycle of eSET + one cycle of standard treatment policy [STP, i.e. eSET in patients <38 years of age with at least one good quality embryo and double embryo transfer (DET) in the remainder of patients] + one cycle of DET have an ICER of 16 593 compared with three cycles of eSET. Furthermore, three STP cycles have an ICER of 17 636 compared with one cycle of eSET + one cycle of STP + one cycle of DET, and three DET cycles have an ICER of 26 729 compared with three cycles STP.
Our study shows that in patients qualifying for IVF treatment, combining several transfer policies was not cost-effective. A choice has to be made between three cycles of eSET, STP or DET. It depends, however, on society's willingness to pay which strategy is to be preferred from a cost-effectiveness point of view.
Because there is a lack of longitudinal research examining the impact of partner coping in couples experiencing infertility, we know very little about the long-term nature of coping with infertility and how partner coping strategies impact personal, marital and social distress.
Participants were Danish men and women about to start a cycle of assisted reproduction treatment who were followed for a 5 year period of unsuccessful treatments. Multilevel modeling using the actor–partner interdependence model was used to examine the couple as the unit of analysis.
Active and passive avoidance coping strategies were significantly related to increased personal, marital and social distress at the individual and partner level. Meaning-based coping strategies were related to decreases in a woman’s individual distress and her partner’s marital distress.
Partner coping strategies have a significant impact on the other member of the couple over time in men and women undergoing infertility treatments over a 5 year period. Physicians and mental health professionals can educate men and women regarding the ineffectiveness of avoidance coping strategies as well as the beneficial nature of finding new meaning and life goals while experiencing the stress of infertility.
Preconception sex selection for non-medical reasons is a controversial issue in bioethics. Little research has described preferences for preconception sex selection among Arab populations. This study describes the sex preference and interest in employing sex selection techniques among pregnant women in northern Jordan.
A self-reported questionnaire was administered to 600 pregnant women in Irbid, Jordan. 2 test and binary logistic regression were used to examine the factors associated with interest in preconception sex selection.
In general, the interest in using sex selection was low. Women who preferred boys were more likely to be interested in sex selection, if paid for by the couple [odds ratio (OR) = 4.40, 95% confidence interval (CI): 1.75–11.11] or by health insurance (OR = 3.42, 95% CI: 1.94–6.06), or, if feasible, administered through oral medication (OR = 8.84, 95% CI: 5.05–15.63). Women with lower education were more likely to be interested in sex selection, if paid by health insurance (OR = 1.96, 95% CI: 1.10–3.45) and were more likely to believe that sex selection is legal (OR = 1.79, 95% CI: 1.06–2.86). Women who had no boys were more likely to be interested in sex selection, if paid by health insurance (OR = 1.94, 95% CI: 1.10–3.42) or, if feasible, through medication (OR = 3.03, 95% CI: 1.82–5.00).
The majority of participants were not in favor of using preconception sex selection. Those with a preference to have boys, with lower education, and those with an imbalanced family were more likely to be interested in using sex selection technology.
Controlled-rate freezing of ovarian cortical tissue for preservation of fertility among young women facing chemo- or radio-therapy is a widely accepted procedure. To improve the method for cryopreservation of ovarian tissue, particularly the stroma, we carried out a systematic comparison of vitrification versus slow programmed freezing.
Ovarian tissue from 20 women, donated during Caesarean section, was used for parallel comparison of survival and detailed light and electron microscopic (EM) morphology of oocytes, granulosa cells and ovarian stroma after freezing (slow freezing and vitrification), thawing and 24-h culture. Using tissue obtained from the same patient, we compared four cryopreservation protocols and fresh tissue. The cryoprotectants used in slow freezing were 1,2-propanediol (PrOH)-sucrose and ethylene glycol (EG)-sucrose. For vitrification, tissues were incubated for 5 or 10 min in three solutions containing a combination of dimethyl sulphoxide (DMSO), PrOH, EG and polyvinylpyrrolidone (PVP).
Cryopreservation using controlled-rate freezing and vitrification preserved the morphological characteristics of ovarian tissue generally well. As revealed by morphological analysis, particularly EM, the ovarian stroma was significantly better preserved after vitrification than after slow freezing (P < 0.001). The follicles were similarly preserved after all freezing methods.
Vitrification using a combination of PrOH, EG, DMSO and PVP was comparable to slow freezing in terms of preserving follicles in human ovarian tissue. Ovarian stroma had significantly better morphological integrity after vitrification than after controlled-rate freezing.
Cryopreservation of whole ovaries followed by vascular transplantation may improve long-term function in comparison to conventional cryopreservation of ovarian cortex and avascular transplantation. The aim of this study was to assess methods for the evaluation of viability and function of frozen–thawed whole ovaries.
Ewe ovaries were flushed with either cryoprotectant (propandiol: FROZEN-PROH) or Ringer Acetate (FROZEN-RA) followed by slow freezing. Some ovaries were assessed fresh after flushing with Ringer Acetate (FRESH-RA). Assessment was done by light microscopy, biochemical response (cyclic adenosine 3',5'-monophosphate (cAMP) and steroids) during in vitro perfusion with forskolin, viability assay and cell culture.
Microscopy showed well-preserved morphology with the presence of small follicles in all groups before perfusion. Stromal oedema was seen after in vitro perfusion of FROZEN ovaries, and shrunken small follicles were seen only in FROZEN-RA at the end of perfusion. During in vitro perfusion, FRESH-RA ovaries responded with large increase in levels of cAMP after stimulation with forskolin. FROZEN-PROH and FROZEN-RA ovaries exhibited lower production of cAMP. Progesterone concentrations in cell cultures of dispersed ovarian cells were higher in FRESH-RA when compared with FROZEN groups. Addition of hCG to cell cultures resulted in higher progesterone levels in the FROZEN-PROH compared with FROZEN-RA. Cell viability assay showed overall viability of 60–75% with no significant difference between groups.
In vitro perfusion may prove to be a suitable method to test viability and function of frozen–thawed whole ovaries contributing to the optimization of current cryopreservation protocols.
Immune alterations may be involved in the pathogenesis and progression of endometriosis. Dendritic cells (DCs) are potent antigen presenting cells that are highly involved in the initiation of the immune response. The aim of this study was to investigate DC populations in the eutopic and ectopic endometrium of women with endometriosis compared with controls.
Hysterectomy samples were obtained from premenopausal women with (n = 33) and without (n = 28) endometriosis. In addition, paired peritoneal endometriotic lesions and uterine curettings were collected from 32 women with endometriosis. Specimen sections were stained immunohistochemically using antibodies for monoclonal mouse antibodies directed against human CD1a and CD83, which are specific for immature and mature DCs, respectively.
The mean density of endometrial CD1a+ DCs in the basal layer was significantly increased in women with endometriosis compared with controls during the proliferative phase only (P = 0.001). There was a highly significant decrease in the density of endometrial CD83+ DCs in women with endometriosis compared with controls in both layers of the endometrium across all phases of the menstrual cycle (P = 0.001). The density of CD1a+ DCs was significantly increased in peritoneal endometriotic lesions (P = 0.003) and in the surrounding peritoneum (P = 0.001) compared with paired uterine curettings and peritoneum distant from the lesion.
Both CD1a+ and CD83+ DC populations were altered in the eutopic and ectopic endometrium of women with endometriosis compared with controls. Alterations in these cells, which play a crucial role in the coordination of the immune response, may be involved in pain generation and the pathogenesis of endometriosis.
The spermatogonial stem cell (SSC) pool in the testes of non-human primates is poorly defined.
To begin characterizing SSCs in rhesus macaque testes, we employed fluorescence-activated cell sorting (FACS), a xenotransplant bioassay and immunohistochemical methods and correlated our findings with classical descriptions of germ cell nuclear morphology (i.e. Adark and Apale spermatogonia).
FACS analysis identified a THY-1+ fraction of rhesus testis cells that was enriched for consensus SSC markers (i.e. PLZF, GFR1) and exhibited enhanced colonizing activity upon transplantation to nude mouse testes. We observed a substantial conservation of spermatogonial markers from mice to monkeys [PLZF, GFR1, Neurogenin 3 (NGN3), cKIT]. Assuming that molecular characteristics correlate with function, the pool of putative SSCs (THY-1+, PLZF+, GFR1+, NGN3+/–, cKIT–) comprises most Adark and Apale and is considerably larger in primates than in rodents. It is noteworthy that the majority of Adark and Apale share a common molecular phenotype, considering their distinct functional classifications as reserve and renewing stem cells, respectively. NGN3 is absent from Adark, but is expressed by some Apale and may mark the transition from undifferentiated (cKIT–) to differentiating (cKIT+) spermatogonia. Finally, the pool of transit-amplifying progenitor spermatogonia (PLZF+, GFR1+, NGN3+, cKIT+/–) is smaller in primates than in rodents.
These results provide an in-depth analysis of molecular characteristics of primate spermatogonia, including SSCs, and lay a foundation for future studies investigating the kinetics of spermatogonial renewal, clonal expansion and differentiation during primate spermatogenesis.
The aim of this study was to investigate the cellular/biochemical mechanisms by which cisplatin (CIS) causes testicular toxicity. We evaluated the role of inducible nitric oxide synthase (iNOS) expression, mitogen-activated protein kinase (MAPK) and nuclear factor-kappa B (NF-kB) activation in the pathogenesis of testicular damage induced by CIS, and investigated the effects of curcumin (CMN) against CIS-induced testicular injury in rats.
Rats were divided into five equal groups: (1) control, (2) CIS, (3) CMN, (4) CIS + CMN and (5) CIS + corn oil. After the treatment, body and testicular weights, and plasma testosterone levels were observed, along with the biochemical, histopathological and immunohistochemical changes in testes.
Testicular weight, plasma testosterone levels, activities of glutathione peroxidase (GSH-Px) and glutathione (GSH) levels significantly decreased, whereas the level of malondialdehyde (MDA) and nitric oxide (NO) significantly increased with CIS compared with the controls. A significant increase in plasma testosterone levels, GSH levels and GSH-Px activity, and a decrease in MDA and NO levels in testicular tissue were observed with CIS + CMN compared with that with CIS alone. There was marked staining for iNOS, MAPK/p38 and NF-kB/p65 expression with CIS compared with the control and CIS + CMN groups. CIS caused irregular seminiferous tubules, reduction of seminiferous epithelial layers, significant maturation arrest and perivascular fibrosis. CMN administration to CIS-treated rats significantly prevented these histopathologic changes.
MAPK and NF-kB activation have a significant role in CIS-induced testicular toxicity. CMN has a strong potential for use as a therapeutic adjuvant in CIS gonadotoxicity.
Metabolic disturbances are common features of polycystic ovary syndrome (PCOS), which possibly enhance the risk of cardiovascular disease. The aim of this study was to assess the accuracy of lipid accumulation product (LAP) index as a marker of cardiovascular risk in PCOS patients.
Case–control study including 51 PCOS patients aged between 14 and 35 years and 44 body mass index (BMI) and age-matched controls. Measures included the LAP index, homeostasis model assessment (HOMA) index, glucose tolerance and plasma hormones, cholesterols and triglycerides.
LAP index was positively correlated with HOMA index in all subjects (r = 0.70; P < 0.001). Waist circumference (WC) (P = 0.002), HOMA index (P < 0.001) and LAP index (P = 0.035) were higher in PCOS patients than controls. On receiver operating characteristic curve analysis, an LAP index of 34.5 (sensitivity: 84%; specificity 79%) showed a better performance than non-high-density lipoprotein cholesterol, WC or BMI to identify insulin resistance (IR) in all subjects. In PCOS patients, the positive and negative predictive values for LAP ≥ 34.5 were 91 and 74%, respectively, compared with 73 and 61%, respectively, for WC ≥80 cm, and 43 and 20%, respectively, for WC ≥88 cm.
We have confirmed that IR is more common in PCOS than BMI-matched control women. Furthermore, the LAP index, an easily obtainable measure, is associated with HOMA index and an LAP ≥ 34.5 is an additional risk factor for cardiovascular disease in PCOS patients.
Current data suggest that excessive androgen exposure can lead to the development of polycystic ovaries and polycystic ovary syndrome (PCOS). Anti-Müllerian hormone (AMH) levels reflect the number of small antral follicles in the ovaries and are elevated in PCOS. We hypothesized that protracted reduction of circulating androgens and/or insulin resistance would reduce circulating AMH concentrations in women with PCOS.
A prospective, randomized, double-blind 26 week long study was undertaken in 50 women with PCOS. They all received diet and lifestyle counselling, and metformin 850 mg three times daily. Concomitantly, they were randomized to either dexamethasone 0.25 mg daily (n = 25) or placebo (n = 25). Thirty-eight women completed the study. AMH (primary outcome) and other hormone levels were measured at inclusion and after 8 and 26 weeks of treatment.
At baseline in univariate regression analyses, AMH levels associated positively with testosterone levels (P = 0.041) and ovarian volume (P = 0.002). In multivariate regression analyses, AMH associated positively with testosterone P = 0.004), and negatively with dehydroepiandrosterone sulphate (DHEAS) (P = 0.001) and C-peptide levels (P = 0.020). Circulating AMH concentrations were unaffected by 6 months of lifestyle counselling with metformin and placebo treatment. AMH levels were also unaffected by 6 months of androgen suppression with dexamethasone in addition.
AMH levels in untreated PCOS women associated positively with testosterone, and negatively with DHEAS and C-peptide levels. Six months of androgen suppression by either metformin or low-dose dexamethasone treatment failed to influence circulating AMH levels.
To determine the optimal daily dose of intranasal hormone therapy (HT) in order to achieve adequate endometrial protection.
Multicentre, double-blind/double-dummy randomized controlled trial (Phase III) was conducted in 1741 women to compare a daily intranasal dose of 350 µg 17β-estradiol (E2) together with 50, 175 or 550 µg norethisterone (NET) with the oral administration of 2 mg E2 and 1 mg NET, over a period of 52 weeks. An endometrial biopsy was performed at the end of HT use.
Most women (73–86%) had an ‘atrophic and/or inactive’ endometrium. Lower doses of NET were associated with a higher incidence of ‘proliferative’ endometrium. The incidence of vaginal bleeding decreased with time. During the last 4 months of the study, 88.1% of women using the highest dose of NET were in amenorrhoea when compared with 71.7% using the oral comparator (difference 16.5%; 95% confidence interval: 10.9–22.0%) (P < 0.001). Premature discontinuation rates were in the range of 12–17% for the three nasal regimens and 22% for the oral comparator.
HT using a fixed intranasal dose of 350 µg E2 combined with 550 µg NET is a safe regimen, in relation to 1 year endometrial safety. This regimen is associated with less vaginal bleeding when compared with an oral comparator using 2 mg E2 and 1 mg NET.
Corticotropin-releasing hormone (CRH) and its receptors have been identified in female reproductive tissues. CRH regulates follicular maturation, ovulation, luteolysis and steroidgenesis. A CRH-related peptide stresscopin (SCP), or urocortin III (Ucn3), has recently been identified, but its functions in the ovary remain to be elucidated. In the present study, we investigated the effects of SCP/Ucn3 on progesterone production in cultured human granulosa-lutein cells.
The presence of SCP/Ucn3 and CRH type-2 receptor (CRHR2) in cultured granulosa-lutein cells from 21 infertile women (aged 22–36 years) was examined by RT–PCR and immunocytochemistry. The concentration of SCP/Ucn3 in follicular fluid, human serum and culture medium was examined by radioimmunoassay. Progesterone production by cultured granulosa-lutein cells treated with SCP/Ucn3 was examined by enzyme-linked immunosorbent assay.
SCP/Ucn3 and CRHR2 mRNAs and proteins were expressed in granulosa-lutein cells. SCP/Ucn3 was detected in culture media of granulosa-lutein cells and follicular fluid. Treatment of cultured granulosa-lutein cells with 0.1, 1.0 or 10 nM SCP/Ucn3 decreased progesterone secretion when compared with untreated control (all P < 0.05). Concomitant treatment with the CRHR2 antagonist antisauvagine-30 counteracted the inhibitory effects of SCP/Ucn3 on progesterone secretion, suggesting a mediatory role of CRHR2.
The present results suggest that the SCP/CRHR2 system is present in human ovaries and treatment with SCP/Ucn3 inhibits progesterone production by cultured granulosa-lutein cells through interaction with CRHR2.
A reduction in the sex ratio at birth has been linked to maternal condition during and before pregnancy. A recent study reported an association between maternal exposure to severe life events and sex ratio at birth using the Danish national register. We attempted to replicate that study using a new Danish cohort.
Mothers of all singleton live births (n = 1.35 million births) in Denmark, between 1 January 1980 and 31 December 2002, were linked to data on their children and partners. The old cohort consisted of babies born between 1980 and 1992 (n = 699 362), whereas the new cohort consisted of babies born between 1993 and 2002 (n = 633 451). We defined exposure as death or serious illness in older children and partners in the first trimester or in the 6 months before conception. Sex ratio at birth was defined as the proportion of male live births.
During the study period, there were 1 349 099 singleton live births (692 870 boys and 656 229 girls). The sex ratio at birth in the new cohort was 0.5134. In the new cohort, prenatal exposure to severe life events was not associated with a reduction in the sex ratio at birth [relative risk = 1.00 (95% confidence interval: 0.95–1.05)].
In the new cohort, we did not find strong evidence that, in a stable western population, prenatal exposure to severe life events is associated with a reduction in the sex ratio at birth.
Understanding of the immune events and mechanisms occurring at the feto–maternal interface is likely to help in understanding the ability of the fetus to survive within the maternal body. Evidence supporting extensive roles of natural killer cells during pregnancy gives rise to a possibility that these NK cells can be mis-regulated and involved in fetal allograft rejection. Killer immunoglobulin-like receptors (KIR) play an important role in regulating the NK cell activity through their activating and inhibiting isoforms. Since there exists a considerable, genetically determined variation in the repertoire of KIR receptors between different individuals, a particular maternal KIR repertoire may predispose to recurrent miscarriages (RMs).
Gene-specific PCR amplification (PCR-SSP) was used to determine the individual KIR genotypes in women experiencing RM and controls.
A higher prevalence of activating KIR genes was seen in patients than in controls. Among women experiencing RM, the BB genotypes were more prevalent (P < 0.0001, OR = 4.4, 95% CI = 2.89–6.69) compared with controls.
Our results indicate that the balance between inhibitory and activating receptor-mediated signals present in natural killer cells is inclined toward a more activating state that may contribute to pregnancy loss.
Common knowledge of over a century has it that monozygotic and dizygotic twinning events occur by unrelated mechanisms: monozygotic twinning ‘splits’ embryos, producing anomalously re-arranged embryogenic asymmetries; dizygotic twinning begins with independent ovulations yielding undisturbed parallel embryogeneses with no expectation of departures from singleton outcomes. The anomalies statistically associated with twin births are due to the re-arranged embryos of the monozygotics. Common knowledge further requires that dizygotic pairs are dichorionic; monochorionicity is exclusive to monozygotic pairs. These are fundamental certainties in the literature of twin biology. Multiple observations contradict those common knowledge understandings. The double ovulation hypothesis of dizygotic twinning is untenable. Girl–boy twins differ subtly from all other humans of either sex, absolutely not representative of all dizygotics. Embryogenesis of dizygotic twins differs from singleton development at least as much as monozygotic embryogenesis does, and in the same ways, and the differences between singletons and twins of both zygosities represent a coherent system of re-arranged embryogenic asymmetries. Dizygotic twinning and monozygotic twinning have the same list of consequences of anomalous embryogenesis. Those include an unignorable fraction of dizygotic pairs that are in fact monochorionic, plus many more sharing co-twins’ cells in tissues other than a common chorion. The idea that monozygotic and dizygotic twinning events arise from the same embryogenic mechanism is the only plausible hypothesis that might explain all of the observations.
]]>Results of assisted reproductive techniques from treatments initiated in Europe during 2005 are presented in this ninth report. Data were mainly collected from existing national registers.
From 30 countries, 923 clinics reported 418 111 treatment cycles including: IVF (118 074), ICSI (203 329), frozen embryo replacement (79 140), oocyte donation (ED, 11 475), preimplantation genetic diagnosis/screening (5846) and in vitro maturation (247). Overall, this represents a 13.6% increase since 2004, partly due to inclusion of 28 417 cycles from Turkey. European data on intrauterine insemination using husband/partner's semen (IUI-H) and donor semen (IUI-D) were reported from 21 countries and included 128 908 IUI-H and 20 568 IUI-D cycles.
In 16 countries where all clinics reported to the IVF register, 1115 cycles were performed per million inhabitants. For IVF, the clinical pregnancy rates per aspiration and per transfer were 26.9% and 30.3%, respectively. For ICSI, the corresponding rates were 28.5% and 30.9%. After IUI-H, the clinical pregnancy rate was 12.6% per insemination in women <40. After IVF and ICSI, the distribution of transfer of one, two, three and four or more embryos was 20.0%, 56.1%, 21.5% and 2.3%, respectively. Huge differences exist between countries. The distribution of singleton, twin and triplet deliveries after IVF and ICSI was 78.2%, 21.0% and 0.8%, respectively. This gives a total multiple delivery rate of 21.8% compared with 22.7% in 2004 and 23.1% in 2003. In women <40 years of age, IUI-H was associated with a twin and triplet pregnancy rate of 11.0% and 1.1%, respectively.
Compared with earlier years, there was an increase in the reported number of ART cycles in Europe. Although fewer embryos were transferred per treatment, there was a marginal increase in pregnancy rates and a reduction in multiple deliveries.
There is no systematic study on coiled sperm in semen, although they are commonly observed. This work characterizes coiled sperm in infertile men to understand the clinical implications and investigate the possible cause by osmotic swelling.
Coiled sperm in semen from 439 infertile patients were quantified and their ultrastructure examined by electron microscopy. Hypo-osmotic swelling (HOS) and demembranation tests were performed to elucidate the nature of the coiling.
Semen from patients contained overall 3% of sperm with head-in-coil (HIC) and 8% other coiled forms, with 12% of patients having 20% or more such sperm. The percentage of coiled sperm (but not HIC) was correlated with age (R = 0.26, P = 0.003) and the epididymal secretory marker neutral -glucosidase (R = 0.16, P < 0.001), and associated with heavy smoking and varicocele. Electron microscopy revealed coiling of tail filaments within the plasma membrane, resembling HOS. Some seminal coiled sperm and most sperm freshly coiled upon HOS could be opened by demembranation, while those that could not be opened were probably fixed in position by oxidation, which occurred more frequently in patients than semen donors.
Sperm coiling in semen is common and independent of sperm quantity or hormonal status. Whereas HIC may have a genetic background, other coiled forms may be associated with a hostile endogenous milieu in the epididymis that causes swelling.
This study investigates whether dietary patterns, substantiated by biomarkers, are associated with semen quality.
In 161 men of subfertile couples undergoing in vitro fertilization treatment in a tertiary referral clinic in Rotterdam, the Netherlands, we assessed nutrient intakes and performed principal component factor analysis to identify dietary patterns. Total homocysteine (tHcy), folate, vitamin B12 and B6 were measured in blood and seminal plasma. Semen quality was assessed by sperm volume, concentration, motility, morphology and DNA fragmentation index (DFI). Linear regression models analyzed associations between dietary patterns, biomarkers and sperm parameters, adjusted for age, body mass index (BMI), smoking, vitamins and varicocele.
The ‘Health Conscious’ dietary pattern shows high intakes of fruits, vegetables, fish and whole grains. The ‘Traditional Dutch’ dietary pattern is characterized by high intakes of meat, potatoes and whole grains and low intakes of beverages and sweets. The ‘Health Conscious’ diet was inversely correlated with tHcy in blood (β = –0.07, P = 0.02) and seminal plasma (β = –1.34, P = 0.02) and positively with vitamin B6 in blood (β = 0.217, P = 0.01). An inverse association was demonstrated between the ‘Health Conscious’ diet and DFI (β = –2.81, P = 0.05). The ‘Traditional Dutch’ diet was positively correlated with red blood cell folate (β = 0.06, P = 0.04) and sperm concentration (β = 13.25, P = 0.01).
The ‘Health Conscious’ and ‘Traditional Dutch’ dietary pattern seem to be associated with semen quality in men of subfertile couples.
In a previous study on severely infertile men, we observed alterations in the number of meiotic crossovers; however, it is unknown if these men also show alterations in the position of crossovers.
Spermatocytes from 15 men (5 control men and 10 infertile men) were immunostained to observe the synaptonemal complex and MLH1 foci, which localize to sites of crossovers. Fluorescent in situ hybridization was performed to identify chromosomes 13, 18 and 21. Chromosome bivalents were separated into those with single and double crossover configurations, and the distribution of MLH1 foci along each chromosome arm was calculated. The inter-focal distances on chromosome 13 and 18 bivalents with double crossovers were also calculated.
Four of the infertile men displayed an altered MLH1 distribution on at least one of the chromosome arms studied. Of these four men, two displayed reduced rates of meiotic recombination. Only one man displayed an abnormality in crossover interference, with inter-focal distances reduced on chromosome 13 bivalents.
Recombination defects in infertile men may include alterations in the number of crossovers, the position of crossovers or both. Alterations in both the number and position of crossovers may increase the risk of aneuploid sperm in infertile men.
Cyclophosphamide (Cy), a widely used anticancer drug, is associated with significant testicular damage and sterility. Co-administration of the immunomodulating compound AS101 during chemotherapy treatments was previously shown to protect organs against cytotoxic damage, without attenuating the drug’s anticancer effect. In this animal study, we investigated the effect of AS101 on testicular damage, sperm DNA damage and infertility induced by Cy. Akt and glycogen synthase kinase-3β (GSK-3β) phosphorylation were investigated as a possible chemoprotective mechanism.
Mature male mice, 10 in each group, were injected intraperitoneally with 200 mg/kg Cy once a week for 5 weeks, with or without concurrent treatment with 10 µg per mouse AS101 three times per week. Damage to testicular tubules and sperm production was determined, sperm chromatin damage was analyzed and fertility was gauged. Akt and GSK-3β phosphorylation were evaluated.
Co-treatment with AS101 during the course of Cy administration significantly reduced the percentage of damaged seminiferous tubules (76.0 ± 10.8% versus 40.3 ± 2.6%), and reduced sperm DNA fragmentation (%DFI) from 44.7 ± 1.0% to 25 ± 6.5%. Co-treatment with AS101 also partially protected against the decrease in numbers of impregnated females and litter size. AS101 increased Akt and GSK-3β phosphorylation.
Our results indicate that AS101 can significantly protect against Cy-induced testicular damage and sperm DNA damage, probably by acting through Akt/GSK-3β phosphorylation.
First trimester screening (FTS) for Down syndrome combines measurement of nuchal translucency, free beta-human chorionic gonadotrophin and pregnancy-associated plasma protein-A (PAPP-A). The aim of this study was to undertake a detailed analysis of FTS results in singleton pregnancies conceived using assisted reproductive technologies (ART) and non-ART pregnancies.
A record linkage study compared outcomes in 1739 ART-conceived and 50 253 naturally conceived pregnancies.
Overall, significantly lower PAPP-A levels were detected in ART pregnancies (0.83 multiples of median, MoM) than in controls (1.00 MoM) (t-test P < 0.001). This difference remained after excluding complicated pregnancies. Analysis of factors affecting PAPP-A levels suggested fresh compared with frozen embryo transfers and use of artificial cycles compared with natural cycles for frozen transfers were associated with lower values. The adjusted odds ratio (AdjOR) for receiving a false-positive result was 1.71 (95% CI 1.44–2.04; P < 0.001) for ART pregnancies compared with non-ART pregnancies, and this leads to a higher AdjOR (1.24, 95% CI 1.03–1.49; P = 0.02) for having a chorionic villous sampling (CVS) or amniocentesis.
ART pregnancies have reduced FTS PAPP-A levels leading to an increased likelihood of receiving a false-positive result and having a CVS/amniocentesis. Lower PAPP-A may reflect impairment of early implantation with some forms of ART.
Soluble human leucocyte antigen-G (sHLA-G) is secreted by extravillous trophoblast (EVT) and has roles in regulating immune cells within the decidua. HLA-G expression on EVT increases as they approach uterine spiral arteries and we have suggested that sHLA-G may be important in the remodelling of these vessels. The autocrine role of sHLA-G in regulating trophoblast function at this critical phase has not been studied. We aimed to investigate the effects of sHLA-G on trophoblast motility, invasion and survival.
The human EVT line, SGHPL-4, was stably transfected to over-express sHLA-G (SGHPL-4sG1). Motility and apoptosis were assessed by time–lapse microscopy. Cells were cultured on microcarrier beads embedded in fibrin gels to assess invasion. The effect of sHLA-G expression on motility, invasion and apoptosis in response to stimulation with either hepatocyte growth factor (HGF) or epidermal growth factor (EGF) was determined.
There was no difference in the motility of either SGHPL-4 cells or SGHPL-4sG1 cells in the absence of stimulation. However, sHLA-G inhibited HGF-induced EVT motility. HGF- and EGF-induced invasions were significantly inhibited in SGHPL-4sG1 compared with SGHPL-4 cells. Increased expression of HLA-G had no significant effect on tumour necrosis factor (TNF)-/actinomycin-induced apoptosis.
Growth factor-stimulated trophoblast motility and invasion are regulated by sHLA-G, indicating a novel autocrine role. The inhibition of trophoblast invasion at the spiral artery may be important to allow interactions leading to vascular remodelling.