BACKGROUND

Grafting of testicular tissue into immunodeficient mice has been used to differentiate the neonatal testes from different animal species up to the level of complete spermatogenesis; however, this approach has not been successful for human testicular tissue. The aim of this study was to evaluate the capacity for differentiation of infant human testicular tissue grafts.

METHODS AND RESULTS

Testicular tissue from a 3-month-old patient with testicular cancer was grafted into immunodeficient nude mice. At the time of grafting, A spermatogonia were the only germ cells present in the testicular tissue. B spermatogonia and first spermatocytes were observed at 7 months and 1 year after grafting, respectively. Positive immunostaining with antibodies against BOULE and CDC25A suggested that spermatocytes in the graft were not arrested but in meiosis. Furthermore, ultrastructural and immunohistochemical analyses showed that the onset of both Sertoli cell maturation and partial differentiation of Leydig cells preceded the appearance of spermatocytes. Differentiation of testicular cells was accelerated compared with in vivo development.

CONCLUSIONS

Spermatogenesis in the xenograft of infant human testicular tissues proceeded successfully from the stage of spermatogonial stem cells until pachytene spermatocyte formation. The differentiation of Sertoli cells and Leydig cells was reproduced in a manner similar to that in normal testicular development. Grafting of infant human testicular tissue may be a powerful tool to examine the early period of human spermatogenesis and may pave the way for fertility preservation among infant patients.

BACKGROUND

Obesity has been identified as a risk factor for spontaneous miscarriage although the mechanism is unclear. The purpose of this study is to better understand the effect of obesity on early pregnancy success by examining the cytogenetic results of miscarriages in women with normal and elevated body mass index (BMI).

METHODS

We conducted a retrospective case–control study in an academic infertility practice. Medical records of women ages <40 years with first trimester missed abortion (n = 204), who underwent dilatation and curettage between 1999 and 2008, were reviewed for demographics, BMI, diagnosis of polycystic ovary syndrome (PCOS) and karyotype analysis. ² and Student's t-test analysis were used for statistical analysis, with P < 0.05 considered significant.

RESULTS

A total of 204 miscarriages were included, from women with a mean age of 34.5 years. The overall rate of aneuploidy was 59%. Women with BMI ≥ 25 kg/m² had a significant increase in euploid miscarriages compared with women with lower BMI (P = 0.04), despite a similar mean age (34.4 years for both).

CONCLUSIONS

We found a significant increase in normal embryonic karyotypes in the miscarriages of overweight and obese women (BMI ≥ 25). These results suggest that the excess risk of miscarriages in the overweight and obese population is independent of embryonic aneuploidy. Further studies are needed to assess the impact of lifestyle modification, insulin resistance and PCOS on pregnancy outcomes in the overweight and obese population.

BACKGROUND

Routes of trophoblast invasion seem to be clear, whereas specific invasive pathways need further elucidation. Extravillous trophoblasts (EVTs) transform spiral arteries to guarantee appropriate blood flow to the placenta in the second trimester. Embryo nutrition during the first trimester is thought to be histiotrophic, whereas proof that EVTs also invade uterine glands is lacking. We developed novel three-dimensional confrontation co-culture models to elucidate invasion of EVTs into uterine glands.

METHODS

First trimester decidua parietalis and placental villous explants were directly confronted and co-cultured for 72 h, or confronted indirectly after 72 h pre-culture for re-epithelialization of decidua pieces. Cryosections were stained by immunohistochemistry or immunofluorescent/immunohistochemical double labelling and compared with first trimester placentation sites in situ.

RESULTS

EVTs deeply invaded decidual tissues in direct confrontation assays and were found between the decidual epithelial cells and epithelial basement membrane. EVTs were also detected in the decidual stroma in direct proximity to glands, sometimes even replacing glandular epithelial cells. Similar observations were made in sections from the first trimester decidua/placental bed. In the invaded parts of sections of decidua basalis, 55% ± 7% (mean ± SEM; n = 10, range 6–11 weeks) of glandular cross sections were associated with or infiltrated by EVTs.

CONCLUSIONS

Using novel confrontation co-culture assays, a potential new route of EVT invasion was detected. EVTs appear to break through the basement membrane of uterine glands to open their lumen towards the intervillous space. These data support the hypothesis of histiotrophic nutrition of the embryo prior to onset of maternal blood flow within the placenta.

BACKGROUND

Extravillous trophoblast (EVT) cell invasion of uterine decidua and the inner third of myometrium is critical for successful pregnancy. Many decidual factors are likely to play a role in regulating this process. We have previously shown that cytokines, known to be produced by uterine natural killer (uNK) cells, such as TNF-, TGF-β1 and IFN- inhibit EVT invasion. We therefore hypothesized that supernatants from purified uNK cells would inhibit EVT invasion.

METHODS AND RESULTS

Total unfractionated decidual cell supernatants from 8 to 10 weeks gestation increased EVT invasion from placental villous explants, although uNK cell supernatants from 8 to 10 weeks gestation had no effect. In contrast, both total decidual and uNK cell supernatants from 12 to 14 weeks gestation stimulated EVT invasion. MMP-2, uPA, PAI-1 and PAI-2 levels did not differ under any of the conditions tested, whereas MMP-9 levels were increased in the presence of both total decidual and uNK cell supernatants from both gestational age groups. There was a decrease in the level of EVT apoptosis in the presence of uNK cell supernatant from 12 to 14 weeks, but not 8–10 weeks, gestation.

CONCLUSIONS

Decidual uNK cell supernatants from 12 to 14 weeks gestational age stimulated EVT invasion, potentially by increasing MMP9 levels and reducing apoptosis. Total decidual cell isolates stimulated EVT invasion at both gestational ages investigated, potentially reflecting the complex nature of these cell culture supernatants.

BACKGROUND

Thymic stromal lymphopoietin (TSLP) is a novel cytokine that triggers the dendritic cell-mediated T_H2 response and regulatory T cell expansion. The aim of this study is to evaluate whether TSLP and TSLP receptor (TSLPR) are expressed in primary human extravillous trophoblast (EVT), how proimflammatory cytokines (tumor necrosis factor (TNF)-, IL-1β), T_H2 and T_H3 cytokines (IL-4, TGF-β) and pregnancy-associated hormones regulate TSLP production by EVT and whether the SLP–TSLPR interaction affects the biological behavior of trophoblsts.

METHODS

We assessed TSLP mRNA and protein expression by real-time RT–PCR, ELISA and immunochemistry, respectively. We further investigated effects of TSLP on the proliferation and invasion of trophoblast cells in vitro.

RESULTS

The primary EVTs constitutively expressed TSLP and TSLPR. IL-4 and TNF- or pregnancy-associated hormones result in a significant increase in TSLP mRNA expression and protein release from EVT, and TSLP promotes primary EVT proliferation and invasion in vitro.

CONCLUSIONS

This study has demonstrated that the first-trimester human trophoblast cells express TSLP and TSLPR, that cytokine milieu which mimics the maternal–fetal interface modulates expression of TSLP in trophoblast and that TSLP stimulates trophoblast proliferation and invasion. This suggests that TSLP plays an important role in human EVT invasion and placentation in human early pregnancy.

BACKGROUND

Home use of misoprostol for termination of pregnancy is still controversial in many countries. Acceptability of home use of misoprostol has been investigated in pregnancies below 49 days gestation. In this study, we aimed to assess efficacy, feasibility and acceptability of home use of vaginal misoprostol for medical termination of pregnancy at 50–63 days compared with gestation of below 50 days among women who chose to administer misoprostol at home.

METHODS

In this prospective study, mifepristone 200 mg was given in hospital under nursing supervision in the university hospital outpatient family planning clinic. Women self-administered misoprostol 800 µg vaginally 36–48 h later at home. Follow-up was 2 weeks later. Women with gestation of 50–63 days on the day of mifepristone administration were compared with women with gestation of below 50 days. Efficacy and feasibility were assessed by review of patient records and questionnaires. Acceptability was assessed using questionnaires where women reported on future choice of abortion method were they to have another abortion.

RESULTS

Among the 2992 women who had a medical abortion during the study period, 395 women chose to administer misoprostol at home and were included in the study. A total of 203 women were below 50 days gestation and 192 were between 50 and 63 days gestation. Efficacy was 97.5% and did not differ between the groups. Surgical intervention was needed in 10 patients, of whom four were in the lower gestational band (P = 0.36). No serious adverse events or blood transfusions were reported. Preference for home administration of misoprostol, were they to have another induced abortion in the future, was high, 92.3 and 86.6% respectively, and did not differ between the groups (P = 0.097). Need for extra analgesia significantly influenced women's experiences in both gestational groups.

CONCLUSIONS

Medical abortion with mifepristone followed by home administration of vaginal misoprostol is safe and highly acceptable also to women with gestational length of 50–63 days as compared with shorter gestations. Efficacy, acceptability and preference for future place of administration of misoprostol, were women to have another abortion, did not differ between women with gestation below 50 days or between 50 and 63 days.

BACKGROUND

The levonorgestrel-releasing intrauterine system (LNG-IUS) induces amenorrhoea, and its effect on bone mineral density (BMD) may constitute a concern. This study evaluated BMD in long-term users of the LNG-IUS or intrauterine device (IUD).

METHODS

BMD was evaluated at the midshaft of the ulna and ultra-distal radius using dual-energy X-ray absorptiometry in 37 women at 7 or 10 years of use following placement of a second LNG-IUS. The groups were paired for duration of use, age, body mass index (BMI), ethnicity and number of pregnancies.

RESULTS

The mean age of both LNG-IUS and IUD users at the 7th and 10th year was ~34 and 38 years, respectively. Mean BMI was ~25 in both groups, increasing to ~26 at the 10th year. Amenorrhoea occurred in 51.4 and 91.9% of LNG-IUS users at the 7th and 10th year, respectively. Estradiol levels in LNG-IUS users were normal at both evaluations. There were no differences in BMD (g/cm²) at the midshaft of the ulna nor ultra-distal radius between LNG-IUS and IUD users or between the 7th and 10th years of use in LNG-IUS users. A Z-score below –2SD at the ultra-distal radius was observed in only one LNG-IUS user and in none of the IUD users at the 10th year. Higher BMI and BMD at the seventh year and amenorrhoea were predictors of higher BMD at the 10th year.

CONCLUSIONS

BMD at the midshaft of the ulna and ultra-distal radius in LNG-IUS users were similar to that of IUD users and remained unchanged between the 7th and the 10th years of use.

BACKGROUND

We have attempted to identify structural, physiological and other targets on human sperm vulnerable to the spermicidal action of two novel series of non-detergent molecules, reported to irreversibly immobilize human sperm in <30 s, apparently without disrupting plasma membrane.

METHODS

Three sperm samples were studied. Scanning and transmission electron microscopy were used to assess structural aberrations of sperm membrane; plasma membrane potential and intracellular pH measurements (fluorometric) were used to detect changes in sperm physiology; reactive oxygen species (ROS, fluorometric) and superoxide dismutase activity (colorimetric) were indicators of oxidative stress; and sperm dynein ATPase activity demonstrated alterations in motor energy potential, in response to spermicide treatment. Post-ejaculation tyrosine phosphorylation of human sperm proteins (immunoblotting) was a marker for functional integrity.

RESULTS

Disulfide esters of carbothioic acid (DSE compounds) caused complete sperm attenuation at ≥0.002% concentration with hyper-polarization of sperm membrane potential (P < 0.001), intracellular alkalinization (P < 0.01), ROS generation (P < 0.05) and no apparent effect on sperm (n = 150) membrane structure. Isoxazolecarbaldehyde compounds required ≥0.03% for spermicidal action and caused disrupted outer acrosomal membrane structure, depolarization of membrane potential (P < 0.001), intracellular acidification (P < 0.01) and ROS generation (P < 0.01). Detergent [nonoxynol-9 (N-9)] action was sustainable at ≥0.05% and involved complete breakdown of structural and physiological membrane integrity with ROS generation (P < 0.001). All spermicides caused functional attenuation of sperm without inhibiting motor energetics. Unlike N-9, DSE-37 (vaginal dose, 200 µg) completely inhibited pregnancy in rats and vaginal epithelium was unchanged (24 h,10 mg).

CONCLUSIONS

The study reveals a unique mechanism of action for DSE spermicides. DSE-37 holds promise as a safe vaginal contraceptive.

CDRI Communication No. 7545.

BACKGROUND

Adenomyosis is rarely diagnosed before hysterectomy and commonly coexists with uterine leiomyomas. The objective of this study was to identify distinct features of a concurrent diagnosis of adenomyosis in women with uterine leiomyomas.

METHODS

We conducted a case–control study of women undergoing hysterectomy with a histologic diagnosis of both adenomyosis and leiomyomas and women with uterine leiomyomas but no adenomyosis. A retrospective medical record review of hospital and ambulatory records was performed to ascertain sociodemographic and anthropometric variables, as well as to confirm intraoperative and pathologic findings.

RESULTS

Our study sample comprised 255 patients, 85 women with adenomyosis and leiomyomas and 170 women with only leiomyomas. In multivariable logistic regression analyses, women with adenomyosis and leiomyomas were more likely to have more pelvic pain [odds ratio (OR) 3.4, 95% confidence interval (CI) 1.8–6.4], have less fibroid burden (OR per doubling in fibroid size 0.6, 95% CI 0.5–0.8), were more likely to be parous (OR 3.8, 95% CI 1.4–10.5) and have lower body mass index (OR per 5 unit increase in BMI 0.8, 95% CI 0.6–1.0) when compared with women with leiomyomas alone.

CONCLUSIONS

Women undergoing hysterectomy with both adenomyosis and leiomyomas have a number of different clinical features compared with women with only leiomyomas at the time of hysterectomy. Women with substantial pain despite a smaller fibroid burden may be more likely to have concomitant adenomyosis.

BACKGROUND

Complete hydatidiform mole (CHM) is a high-risk pregnancy for gestational trophoblastic neoplasia (GTN). Patients with CHM have a 10–30% chance of trophoblastic sequelae. CHM includes androgenic homozygous (monospermic) and androgenic heterozygous (dispermic) moles. It is controversial whether the risk of GTN is higher with heterozygous than with homozygous CHM. A prospective cohort study was conducted to assess risk of GTN in homozygous and heterozygous CHM using short tandem repeat (STR) polymorphisms, and a meta-analysis of previous reports.

METHODS

Twenty-eight consecutive molar pregnancies were evacuated and followed by regular hCG measurements to detect GTN. Persistent GTN was diagnosed according to the International Federation of Gynecology and Obstetrics 2000 system. Cytogenesis of the mole was determined by STR polymorphisms of molar tissue and parental blood. A meta-analysis of the GTN rate from previous reports was conducted using Mantel–Haenszel methods.

RESULTS

Of 28 molar pregnancies, 24 were homozygous and three were heterozygous CHM. The remaining mole was diandric triploidy (a partial hydatidiform mole). Of the 24 homozygous CHMs, six (25%) cases developed GTN and received chemotherapy. Meanwhile, all three cases (100%) of heterozygous mole developed GTN and needed chemotherapy. The GTN risk was higher in heterozygous (P = 0.029, Fisher's exact test) than homozygous moles. A systematic review revealed only five previous reports (with more than 15 cytogenetically diagnosed cases), and the pooled relative risk of persistent GTN for heterozygous mole was not significant (odds ratio, 2.0; 95% confidence interval, 0.98–4.07).

CONCLUSIONS

Heterozygous CHM had a higher risk for GTN than homozygous CHM.

BACKGROUND

Recent advancement of minimum volume vitrification methods has resulted in a dramatic increase in the efficiency of the process. The aim of this study was to estimate the cumulative reproductive outcome of a cohort of infertile couples undergoing ICSI and oocyte vitrification in restrictive legal conditions, where only a limited number of oocytes could be inseminated per cycle and embryo selection and cryopreservation were forbidden.

METHODS

In this prospective longitudinal cohort study, the cumulative ongoing pregnancy rates obtained by the insemination of fresh and vitrified oocytes from the same cohort were calculated as primary outcome measures. Moreover, the effect of basal and cycle characteristics on clinical outcomes were assessed.

RESULTS

Between September 2008 and May 2009, 182 ICSI cycles were performed where oocyte vitrification was possible. A total of 104 first and 11 second oocyte warming cycles were then performed in non-pregnant patients of the same cohort. The overall ongoing pregnancy rates obtained in the fresh, and first and second warming cycles were 37.4, 25.0 and 27.3%, respectively. The overall cumulative ongoing clinical pregnancy rate observed per stimulation cycle was 53.3%. Maternal age was the only characteristic found to influence the reproductive outcome, with an inverse correlation between the age >40 and the ongoing pregnancy rates (P = 0.04, by Cox regression analysis).

CONCLUSIONS

High cumulative ongoing pregnancy rates can be obtained with transfers of embryos derived from fresh and cryopreserved oocytes in a typical infertile population. Female age significantly affects outcomes in this system.

BACKGROUND

The number of childhood cancer survivors has dramatically increased and consequently, an increasing number of survivors may now wish to conceive. Recently, several studies have described that previous treatment with abdominal radiotherapy may increase the risk of adverse pregnancy outcome.

METHODS

We conducted a retrospective single centre cohort study of childhood cancer survivors with a singleton live birth between January 2000 and December 2005. Pregnancy outcome was compared with data from the Netherlands Perinatal Registry, a nationwide database of pregnancy outcome parameters of all births in the Netherlands registered by midwives, obstetricians and paediatricians.

RESULTS

Data were available on 40 survivors and 9031 controls. Median age at diagnosis was 6.9 years (range 0.1–16.8 years). The median interval between diagnosis and date of delivery was 21.6 years (range 7.4–36.1 years). In the whole cohort, pregnancy outcome was not different between survivors and controls. However, survivors treated with abdominal radiotherapy delivered preterm and had post-partum haemorrhage (mean gestational age in survivors = 34.9 versus 39.2 weeks in controls, P = 0.001; 33% in survivors versus 5% in controls, P = 0.007, respectively). The offspring of survivors had normal birthweight after adjustment for gestational age (mean birthweight in offspring of survivors 2503 versus 1985 g; P = 0.22).

CONCLUSION

Childhood cancer survivors irradiated to the abdomen have an earlier delivery and higher incidence of post-partum haemorrhage. This stresses the need for close monitoring of the delivery, including inpatient perinatal care, in this group of childhood cancer survivors.

BACKGROUND

The prevention of multiple pregnancies remains a major challenge in patients treated with ovarian stimulation prior to intrauterine insemination (IUI). The pilot study presented here investigates whether multiple pregnancies can be minimized by a microscopically confirmed aspiration of oocytes from supernumerary follicles immediately before intrauterine insemination and evaluates the benefit of concomitant excess oocyte cryopreservation for future use.

METHODS

Thirty-four aspirations of supernumerary follicles were performed immediately prior to IUI in 31 patients undergoing ovarian stimulation. sIUI was only performed if cumulus-oocyte complexes were microscopically observed in the aspirated follicular fluid. All collected mature excess oocytes were cryopreserved using the vitrification technique.

RESULTS

Only four sIUI procedures had to be cancelled due to failed oocyte retrieval or premature ovulation. IUI treatment resulted in a clinical pregnancy rate of 23.5% per cycle. All were singleton pregnancies. A total of 111 oocytes were cryopreserved. Patients with polycystic ovary syndrome (PCOS) had an average of 6.07 oocytes vitrified, whereas patients without PCOS had 1.3 oocytes vitrified per cycle.

CONCLUSION

Microscopically confirmed collection of excess oocytes prior to stimulated IUI reduced cancellation rates, further reduced the risk for multiple pregnancy and may lead to future additional pregnancies because, based on current information, approximately 5% of the vitrified oocytes could potentially establish a pregnancy.

BACKGROUND

The use of GnRH antagonists in IVF treatment has many advantages over agonist long down-regulation, yet its uptake has been hampered by an inability to program the start date for gonadotrophin stimulation so as to minimize weekend oocyte retrievals (ORs). In this study, we retrospectively analyzed whether conducting a strict Monday to Friday OR program impacts on IVF outcomes.

METHODS

A total of 1642 non-programmed IVF antagonist cycles were analyzed to determine if advancing or delaying the OR by 1 day from ‘ideal’ to avoid Saturday or Sunday OR, respectively, had any impact on IVF outcomes. The IVF outcomes of Tuesday to Thursday served as a control as no modification in OR timing was required on these days.

RESULTS

Advancing the OR by 1 day from the ideal resulted in a small but significant decrease in the number of oocytes collected and embryos created. Delaying the OR by 1 day from ideal resulted in a small increase in the number of oocytes collected and embryos created. However, deviation from the ideal day of OR had no significant effect on live birth rates.

CONCLUSIONS

It is possible to safely avoid weekend ORs during GnRH antagonist cycles by simply advancing an ideal Saturday OR to Friday, and delaying an ideal Sunday OR to Monday, without adversely impacting on IVF live birth outcomes.

BACKGROUND

Financing ART is variously regulated in the different countries of Europe. In Germany, coverage of assisted reproduction by statutory health insurances was restricted to 50% in 2004. We conducted a national survey among patients, professionals (physicians and other academics in IVF centres, psychosocial counsellors, medical ethicists, social lawyers, health politicians) and the general public in Germany regarding their opinions on financing ART.

METHODS

Standard questionnaire techniques (paper and pencil interviewing, computer-aided web interviewing, computer-aided telephone interviewing) were used.

RESULTS

The vast majority of all groups supported public coverage of ART. Co-payments by patients were considered appropriate by about one-third of the patients, two-third of the physicians and three quarters of all other groups. According to the respondents, the amount of co-payment should cover 15–25% of the costs, considerably less than what patients actually have to pay (50%). Support for public coverage was strongly correlated with the views (i) of infertility as a disease, (ii) that there is a need for assisted reproduction for infertile couples and (iii) that every human should have the opportunity to have children. The respondents had varying opinions on whether to increase medical insurance premiums in order to cover ART. Reducing services in other areas of health care in favour of reproductive medicine was supported only by the group of reproductive physicians. Financial incentives for oocyte sharing were rejected by most groups as was a money-back guarantee for unsuccessful treatments.

CONCLUSIONS

Experts and the general public in Germany accept moderate co-payments for ART. No clear pattern of opinion emerged regarding the question of how public co-funding should be financed.