Effect of gonadotrophin-releasing hormone and related analogue on human luteal cell function in vitro

The possible direct effect of gonadotrophin-releasing hormone(GnRH) and GnRH agonist (GnRH-A; buserelin) on basal and humanchorionic gonadotrophin (HCG)-stimulated progesterone (P) andcyclic AMP (cAMP) production by cultured human luteal cellswas examined. Luteal cells from the early or mid-luteal phasewere incubated in long-term cultures. They responded to HCGstimulation with a 2- to 3-fold increase in P production anda 2-fold increase in cAMP production. The addition of GnRH (10^–7and 10^–5 M) or GnRH-A (10^–7 and 10^–5 M) tothe medium had no effect on either basal or HCG-stimulated secretion.These results indicate that both GnRH and GnRH-A have no directeffect on human luteal steroidogenesis in vitro.     

Fertilization and early embryology: Granulosa cells from fofficle stimulating hormone-primed monkeys enhance the developmental competence of in-vitro-matured oocytes from non-stimulated rhesus monkeys

The specific aims of this study were to determine if culturewith freshly recovered granulosa cells obtained from folliclestimulating hormone (FSH)-primed versus non-stimulated monkeysduring in-vitro maturation (IVM) improves the meiotic and developmentalcapacity of oocytes from non stimulated macaque monkeys andconfers on them compet ence to develop into blastocysts in vitro.Antral fofficles 1 mm in diameter were dissected from the excisedovaries of 11 non-stimulated rhesus monkeys. Cumulus-enclosedgerminal vesicle-stage oocytes (n=282) were randomly culturedin each of three IVM treatnients: (i) control (no granulosacells), (ii) non-stimulated granulosa cells (5x10⁶ live cells/ml)from non-stimulated monkeys, or (iii) FSH primed granulosa cells(5x10⁶ live cells/ml) from monkeys primed with purified FSHfor 8.5 days. All treatments contained gonadotrophins (5 µg/mlFSH; 10 µg/ml lutein, izing hormone). Oocytes were culturedfor 36–41 h in 25 L drops (one to 10 oocytes per drop)of modified CMRL 1066 medium containing 20% bovIne calf serum,and then inseminated and cultured in the same medium (minusgonadotrophins) until developmental arrest or zona escape. Nuclearmaturation, fertilization and cleavage to the 8-cell stage didnot differ for the three treatment methods. However, developmentto the 9- to 15-cell stage was improved (P0.01) by culture witheither non-stimulated granulosa or FSH-prhned granulosa cellscompared with control. The culture of oocytes with FSH-priinedgranulosa but not non-stimulated granulosa cells enhanced (P0.05)development to the morula stage compared with control (23, 11and 5% respectively). Oocytes cultured with FSH primed granulosacells were more (P0.05) competent to develop into blastocysts(6.5%) than controls (0.5%) or those cultured with non-stiinuhitedgranulosa cells (0.6%). In conclusion, the culture of immatureoocytes from non- stimulated monkeys with fresh granulosa cellsrecovered from FSH-primed monkeys enhanced their competenceto develop into morulae and blastocysts in vitro, and resultedin the first blastocysts produced in vitro from in-vitro matured/in-vitro-fertilizedoocytes of non-stimulated macaques.     

The developmental potential of mouse oocytes matured in serum-free culture conditions

Studies were undertaken to identify serum-free conditions forthe maturation of mouse oocytes in vitro. Oocytes were recoveredfrom the antral follicles of juvenile mice 48 h after injectionwith gonadotrophin and allowed to resume meiosis in modifiedHam's F-10 (mHF-10) medium unsupplemented or supplemented withbovine serum albumin (BSA), fetal calf serum, human pre-ovulatoryserum, human follicular fluid or EDTA. They were inseminated14–16 h later, scored for polar body extrusion after 4–6h with spermatozoa, and transferred to protein-free mHF-10 forfurther development. In-vivo matured ova were inseminated andcultured in parallel as controls. Fertilization and developmentwere scored as two cells 24 h after insemination and blastocysts4 days following insemination respectively. Surprisingly, 41%of oocytes cultured in unsupplemented mHF-10 completed meiosisI, and of those, 50% fertilized; serum supplementation did notimprove maturation or fertilization rates. Although the additionof human follicular fluid to the mHF-10 improved meiosis (69%)and fertilization (68% of eggs with polar bodies) to levelscomparable with the in-vivo control eggs (79 and 66% respectively),BSA supplementation was equally beneficial. Blastocyst developmentvaried, but within each maturation/ fertilization group, thedevelopment from in-vitro matured eggs was comparable with embryosfrom in-vivo matured eggs. In addition, two out of eight 4-cellembryos from oocytes cultured in mHF-10 with BSA and EDTA gaverise to apparently normal pups following transfer to pseudopregnantrecipients. Thus, gonadotrophin-stimulated mouse oocytes cancomplete meiosis and fertilize in culture in the absence ofserum or follicular fluid. Oocytes cultured overnight in mHF-10,supplemented with EDTA and BSA, complete meiosis I, fertilizeand develop to blastocysts at rates comparable with eggs maturedin vivo. Serum-deprived oocytes have the potential to give riseto live offspring.     

Infertility: Higher pregnancy rates with a simple method for Fallopian tube sperm perfusion, using the cervical clamp double nut bivalve speculum in the treatment of unexplained infertility: a prospective randomized study

The object of this study was to evaluate the efficacy of thenewly developed cervical clamp double nut bivalve (DNB) speculumused for Fallopian tube sperm perfusion (FSP) with 4 ml of theinseminate, in comparison with standard intrauterine insemination(IUI) using a volume of 0.5 ml of the inseminate. Couples withunexplained infertility (n = 104), undergoing 202 cycles, wereenrolled in this study. Cycles were assigned randomly to eitherIUI (group A, n = 92) or FSP + DNB speculum^® (group B, n= 110). Ovarian stimulation was achieved using three differentovarian stimulation protocols in both groups. The age and folliculardevelopment of the patients were similar in both groups. Theserum hormonal measurements and the endometrial thickness wasalso similar on the day of human chorionic gonadotrophin (HCG)administration. The mean (± SD) number of motile spermatozoainseminated was 44.83 ± 16.57 x 10⁶ in group A and 42.68± 13.44 x 10⁶ in group B. In group A (IUI), 11 clinicalpregnancies (presence of gestational sac with heart beats) occurred(11.95% per cycle). In group B (FSP + DNB speculum^®) 29clinical pregnancies occurred (26.36% per cycle). These differenceswere statistically significant (P <0.001). The results ofthis study for the treatment of unexplained infertility indicatethat this simple, well tolerated, inexpensive method of usingthe DNB speculum for FSP is more successful than standard IUI.     

Preiinplantation diagnosis: Ultrasound derived indices of follicular blood flow before HCG administration and the prediction of oocyte recovery and preimplantation embryo quality

The principal aim of the study was to relate ultrasound-derivedindices of blood flow in individual follicles on the day of,but before, the administration of human chorionic gonadotrophin(HCG) to the subsequent recovery of oocytes and the productionof preimplantation embryos. Data were obtained from 21 women(aged 29–43 years) with bilateral tubal occlusion, whowere undergoing treatment by in-vitro fertilization (FVF) andembryo transfer. Transvaginal ultrasonography with colour Dopplerimaging and pulsed Doppler spectral analysis were used to measurefollicular volume and derive indices of blood flow. The end-pointsfor each follicle were the volume, peak systolic velocity (PSV),pulsatility index (PI), and the recovery or non-recovery ofan oocyte, the subsequent production or non-production of apreimplantation embryo and the morphological grade of each embryo.A total of 94 follicles were studied; 74 oocytes were recovered(79%) and 40 embryos (33 grade I or II) were produced. Therewere four clinical pregnancies (pregnancy rate 25.0% per transfer,19.0% per patient). There was a significant correlation betweenwhether or not follicular blood flow was detected and whetheror not an oocyte was recovered (P <0.05, x² test). The valuesfor volume and PI were not clinically useful. The PSV (cm/s,mean ± SD) was higher in follicles that were associatedwith the production of an embryo (12.7 ± 5.9) comparedwith those that were not (8.5 ± 5.0; P <0.05, Student'st-est). The probability of producing a grade I or grade II embryowas 75% if the PSV was 10 cm/s. The corresponding value was40% if the PSV was <10 cm/s and 24% if blood flow was notdetected (i.e. PSV <3 cm/s). There was a significant increase(P <0.05, Student's t-test) in the PSV before aspirationin those follicles associated with the subsequent productionof an embryo. We conclude that the value for PSV, before theadministration of HCG, can be used to identify follicles witha high probability of producing an oocyte and a high grade preimplantationembryo. The information may also be used to time the administrationof HCG to achieve the optimum number and quality of embryosfor patient management.     

Fertilization and early embryology: Intracytoplasmic sperm injection of mouse oocytes with 5 mM Ca2+ at different intervals

The objective of this investigation was to determine whetherintracytoplasmic sperm injection (ICSI) can be performed inthe mouse. Metaphase II oocytes were obtained from F1 hybridmice (C57BLx CBA) by i.p. injections of 10 IU pregnant mare'sserum gonadotrophin (PMSG) and human chorionic gonadotrophin(HCG) administered 48 h apart. Oocytes with cumulus oophoruswere retrieved 13–14 h post HCG. Cumulus was dispersedwith 0.1% hyaluronidase. Mouse spermatozoa were obtained fromthe cauda epididymides of males of the same strain. The spermatozoawere processed by the standard swim-up procedure. The harvestedspermatozoa were then incubated for 1.5 h to allow capacitation.Healthy oocytes were injected with 3–4 pi 5 mM Ca²⁺, followedby one live morphologically normal spermatozoon into the cytoplasmat intervals of 0, 0.5, 1, 2 and 3 h. The proportion of 2-cellembryos that developed from oocytes injected with Ca²⁺ and spermatozoaranged between 29.5 and 36.5% in all groups, with no statisticaldifference between treatments. Chromosomal analysis showed thattwo-thirds of the ICSI-derived 2-cell embryos were diploid.The proportion of parthenogenetically activated embryos in theICSI groups was similar to that in the control group (8–10%)which was injected with Ca²⁺ and polyvinyl pyrrolidone only.The proportion of blastocysts that developed in culture fromthe ICSI-derived 2-cell embryos was of the order of 36–42%.Some blastocysts were used for cell number counts. There wasa significant increase in total and inner cell mass counts ofblastocysts in which the spermatozoon was injected at 2 and3 h following Ca²⁺. The remaining blastocysts were transferredto day 3 pseudopregnant mice, of which 33% subsequently becamepregnant. Of the blastocysts transferred, 16–25% developedto term in vivo. No deformities were observed in the pups. Webelieve this is the first report of live-birth following mouseICSI.     

Zona opening with 308 nm XeCl excimer laser improves fertilization by spermatozoa from long-term vasectomized mice

Spermatozoa from long-term vasectomized mice have greatly reducedfertilizing ability in vivo and in vitro, which makes this auseful animal model for male factor infertility. The purposeof this study was to evaluate the 308 nm XeCl excimer laserfor opening the zona pellucida to enhance the fertilizationrate with spermatozoa from vasectomized males. Inseminatingzona-intact (control) oocytes with 5 x 10⁶ spermatozoa/ml resultedin only 6% fertilization and 33.3% development to the blastocyststage; zona-opened oocytes showed significant improvement with31.5% fertilization, 90% cleavage to the 2-cell stage, and 72.2%blastocyst formation. Out of the 130 oocytes in the experimentalgroup, zona ablation was performed successfully on 127 and onlythree were damaged. These results suggest that laser micromanipulationfor assisted fertilization potentially offers a simplified andprecise method for mechanical zona cutting.     

Epidermal growth factor receptors in human corpora lutea during the menstrual cycle and pregnancy

We have demonstrated the presence of epidermal growth factor(EGF) and its receptors in human non-gestational corpora lutea.To determine further the characteristics of EGF receptor binding,we examined 30 human corpora lutea throughout the luteal phaseand during pregnancy. Scatchard plots of EGF binding in 29 ofthe 30 corpora lutea were curvilinear, suggesting negative co-operativity.The mean ± SE of the association constant K_a was (0.9± 0.2) x 10⁹ 1/mol, the dissociation constant K_d was(2.2 ± 0.3) x10^–9 mol/1 and the number of bindingsites (Rt) was (15.8 ± 2.1) x10^–19 mol/µgprotein for non-gestational corpora lutea. The K_d increasedsignificantly in late pregnancies compared to early pregnancies(P = < 0.005), while Rt was significantly higher in termpregnancies than in either early pregnancy (P < 0.01) orthe menstrual cycle (P < 0.001). Corpora lutea atretica (n= 2) and ovarian stroma (n = 6) did not show any EGF bindingactivity. Our findings demonstrate the presence of specificEGF receptors in human corpora lutea of both the menstrual cycleand pregnancy. The changes in EGF binding parameters in earlypregnancy suggest that there may be a relationship between therole of EGF and ovarian steroidogenesis.     

Augmentation by thyroxine of human granulosa cell gonadotrophin-induced steroidogenesis

The objective of this study was to investigate the influenceof thyroid hormone on gonadotrophin-induced oestradiol and progesteronesecretion by human granulosa cells maintained in vitro. Granulosacells were obtained by aspiration of pre-ovulatory folliclesfrom women undergoing assisted reproductive technology. Ovulationinduction was performed with gonadotrophin-releasing hormoneagonist, human menopausal gonadotrophin and human chorionicgonadotrophin. Granulosa cells were maintained in vitro in adefined medium with added insulin. Between 48 and 72 h afterthe initiation of cell culture, oestradiol and progesteronesecretion into the medium was determined for granulosa cellsgrowing in serum-free medium with follicle-stimulating hormone(FSH)/luteinizing hormone (LH) and in serum-free medium withFSH/LH and thyroxine added in a concentration range of 10^–10–10^–7M. All concentrations of thyroxine used produced a statisticallysignificant increase in oestradiol (range 1.18–1.37 timesthe amount with FSH/LH alone) and progesterone (range 1.29–1.51times the amount with FSH/LH alone) secretion.     

Diazepam induces meiotic delay, aneuploidy and predivision of homologues and chromatids in mammalian oocytes

The tranquilizer and anti-convulsant diazepam (DZ) is a suspectedaneugen. In order to assess its aneugenic potential in mammalianoogenesis we exposed in vitro maturing mouse oocytes to thedrug. Spindle formation and cell cycle progression, the behaviourof chromosomes and the distribution of mitochondria were characterizedwith respect to induction of numerical chromosomal aberrations.A concentration of 25 µg/ml DZ induced a pronounced delayin maturation and blocked a high percentage of oocytes in meiosisI. This arrest was partly reversible. Hyperploidy was slightlyincreased in oocytes matured in the presence of 5 µg/mlDZ and became significantly elevated in oocytes matured with25 µg/ml DZ, relative to controls. Concomitantly, DZ inducedspindle aberrations and displacement of chromosomes from theequator, but unlike in mitosis and in male meiosis most oocytesstill possessed bipolar spindles. A significant fraction ofmeiotically delayed, metaphase I-blocked oocytes exposed to25 µg/ml DZ contained univalents. Some DZ-treated oocytesprogressing to meiosis II exhibited one or multiple single chromatids.Precocious chiasma resolution and equational segregation ofchromatids from functional univalents in first anaphase (predivision)may be responsible for this condition, a mechanism also discussedin the aetiology of maternal age-related aneuploidy. DZ disturbedthe spatiotemporal distribution of mitochondria during oocytematuration, possibly by binding to peripheral-type benzodiazepinereceptors on mitochondria, thus affecting the availability ofATP and calcium homeostasis. Blocks in maturation may also relateto binding of DZ to calmodulin. Data suggest that DZ exposesmammalian oocytes to predivision and aneuploidy. Thresholds,long lasting effects of DZ in vivo and sex-specific sensitivitiesin chemically induced aneuploidy of mammalian germ cells arecritically evaluated. ¹Present address: Department of Genetics Wageningen AgriculturalUniversity, 6703 HA Wageningen, The Netherlands ^*To whom correspondence should be addressed. Tel: +49 521 106 4727; Fax: +49 521 106 6015; Email: eiri{at}biologie.uni-bielefeld.de      

Andrology: An auto-controlled study in in-vitro fertilization reveals the benefit of Percoll centrifugation to swim-up in the preparation of poor-quality semen

The efficiency of spermatozoa prepared by swim-up or by Percollcentrifugation was assessed in an in-vitro fertilization programmeon 71 semen samples of a well-defined quality [total numberof type A (WHO criteria) motile spermatozoa]: category I (n= 21) with > 100 x 10⁶, II (n = 31) with 15–100 x 10⁶,III (n = 11) with 5–15 x 10⁶ and IV (n = 8) with <5 x 10⁶ type A motile spermatozoa. Oocytes were inseminated4 h after oocyte retrieval, alternately with spermatozoa derivedfrom swim-up and Percoll preparation. Both selection proceduresresulted in a significantly higher (P < 0.001) percentagemotility as compared to fresh semen. For low-quality samples(III and IV), however, swim-up was more effective in selectinghighly motile (P = 0.004) and morphologically normal spermatozoa(P < 0.05). For high-quality samples, this difference mighthave been masked by introducing a swim-up step to remove Percollparticles. Regardless of the initial sperm quality, the meanfertilization rate was significantly higher (P = 0.003) whenPercoll-treated spermatozoa were used for insemination (51.3versus 37.8%). For semen of groups I and II, no difference infertilization capacity was observed according to the sperm preparationmethod. Despite the lower percentage motility and normal morphologyfor the Percoll compared to the swim-up treatment in groupsIII and IV, fertilizing capacity was significantly (P < 0.001)in favour of this selection method (65.3 versus 26.5% in groupIII, 47.6 versus 11.6% in group IV). Based on these results,it may be concluded that a subgroup of patients exhibiting poorsemen quality can benefit from Percoll semen preparation interms of improved fertilizing capacity.     

Fertilization and early embryology: Cryopreservation of immature mouse oocytes

Mouse oocytes enclosed in cumulus cells were isolated from antralfollicles at the germinal vesicle (GV) stage. They were storedin straws at – 196°C by a conventional mouse embryofreezing method using dimethylsulphoxide (1.5 M) as the cryoprotectant.Overall survival assessed after removal of the cumulus cellswas 93% (299/320). A significantly greater proportion of freshoocytes remained arrested at the GV stage during culture (11versus 1%), but the rate of maturation to metaphase II was notsignificantly different between frozen and fresh oocytes (83versus 74%). The rate of fertilization in vitro was similarfor frozen and fresh oocytes matured in vitro (70 versus 81%)but significantly less than with mature ovulated oocytes (96%).Fertilization of frozen and fresh oocytes arrested after germinalvesicle breakdown was similar (77 versus 95%. No evidence ofparthenogenetic activation was found in the different groupsafter overnight incubation of metaphase II oocytes. Implantationwas similar for embryos derived from fresh and frozen GV-stageoocytes matured in vitro and mature ovulated oocytes, but theloss of embryos after implantation was significantly higherin the in-vitro matured groups (frozen, 40% and fresh, 46% versus24%). The overall survival of oocytes frozen at the GV stagewas 27%. This compares favourably with the estimated overallsurvival of mature oocytes cryopreserved by a similar procedure.We conclude that the increased post-implantation loss is dueto suboptimal conditions for maturation in vitro rather thanfreezing injury.     

Fractured zona oocytes in in-vitro fertilization cycles stimulated with gonadotrophin-releasing hormone analogue and human menopausal gonadotrophin

In order to assess the possible influence of gonadotrophinreleasinghormone analogue and human menopausal gonadotrophin on the occurrenceof fractured zona oocytes (FZOs) in in-vitro fertilization (IVF)treatment cycles, we analysed 267 consecutive cycles in 199patients. In 87 cycles, at least one fractured zona oocyte wasrecovered, and in 180 cycles only intact zona oocytes (IZOs)were recovered. FZOs represented 5.8% of all oocytes retrievedand 14.8% when only cycles with FZOs were considered. Serumoestradiol concentrations were significantly higher at day –3and day –2 (P < 0.02) in cycles yielding at least onefractured zona oocyte compared to IZO cycles (day 0 = retrievalday), and there was a higher incidence of G terminal patternof oestradiol curve (P < 0.01) in cycles with FZOs. The meannumbers of all oocytes retrieved and of mature oocytes weresignificantly higher in FZO than in IZO cycles (P < 0.001).The fertilization rate of mature oocytes was significantly reduced(P < 0.05) in cycles with one or more oocytes with fracturedzonae. There was no significant difference in the number ofembryos transferred, pregnancy and abortion rates in both groups.We conclude that although the occurrence of fractured zona oocytesis a frequent event, it does not affect the overall resultsof our IVF programme. Zona pellucida fragility may be the resultof over-maturation of some oocytes.     

Hexokinase activity in mouse embryos developed in vivo and in vitro

Micro-determination methods were used for quantitative examinationof possible differences in energy metabolism in mouse embryosarising after spontaneous ovulation or after gonadotrophin stimulation.Comparisons of embryonic development in vivo and in vitro werealso made. The relevance of the results to human developmentand their clinical significance are discussed. The enzymaticactivity of hexokinase, phosphofructokinase, glucose 6-phosphatedehydrogenase, malate dehydrogenase and lactate dehydrogenasein individual mouse embryos throughout preimplantation developmentwas evaluated. Hexokinase activity in 1-cell embryos was thelowest by far of the five enzymes measured, and the 0.035 ±0.010 pmol of nicotinamide adenine dinucleotide phosphate hydrogenaseformed/embryo/min was also lower than in any of the somaticorgans examined. Hexokinase activity, unlike the other enzymes,progressively increased in the morulae and blastocyst stagesin embryos obtained either by spontaneous ovulation or via gonadotrophinstimulation. Although there is a significant delay, this increasewas also observed when 2-cell embryos developed in vitro. Increasesin hexikinase activity were observed 68–75 h after humanchorionic gonadotrophin administration in vivo, but after 80–86h in vitro. These increases in vitro were inhibited by the administrationof actinomycin D added to the medium. The results suggest thathexokinase may be a key enzyme synthesized as the zygotic genomeis expressed in preimplantation embryos, and its measurementmay help to assess the quality of embryos developed in vitro.     

Molecular analyses of in vivo hprt mutations in human T-lymphocytes I. Studies of low frequency 'spontaneous' mutants by Southern blots

Fifty wild-type and 164 in vivo-derived hprt mutant T-cell clonesobtained from eight non-mutagen-exposed adult males with mutantfrequency values in the normal range (usually < 10 x 10^–6)were studied by Southern blot analyses to determine frequencyand extent of gross structural alterations in the hprt gene.Sixteen (9.8%) of the mutant clones showed hprt changes. Nosite or type of lesion predominated. Relative frequencies ofgross structural alterations in the recovered hprt mutants didnot differ among the eight individuals, within limits detectableby the study. DNA from 201 of these 214 clones was also studiedwith a T-cell receptor (TCR) ß gene probe as a markerfor independence of in vivo-derived clones. Some clones werealso studied with a TCR gene probe. Ninety-four percent ofwild-type and 89% of the hprt mutants were found to originatefrom independent in vivo precursors. Therefore, most of therecovered hprt mutants in the study were presumably derivedfrom separate in vivo mutations. For non-mutagenized adultswith normal mutant frequencies, in vivo mutant frequencies arethus reasonable approximations of in vivo mutation frequencies,although elsewhere we show that this is not necessarily truefor individuals with grossly elevated mutant frequencies.     

Fertilization and early embrology: Atypical maturation of oocytes of strain I/LnJ mice

The maturation of strain I/LnJ oocytes was compared to oocytesof selected inbred strains. The time of germinal vesicle breakdown(GVB) of I/LnJ oocytes was greatly delayed compared to all otherstrains tested. In addition, 5% of the cumulus cell-enclosedoocytes isolated from antral follicles of I/LnJ mice failedto undergo GVB in vitro and 58% of the oocytes that underwentGVB failed to progress beyond metaphase I. Similar defects inthe progression of meiosis occurred when maturation was stimulatedin vivo by the administration of exogenous gonadotrophins. Whenin-vitro matured metaphase II oocytes were selected for in-vitrofertilization, similar percentages of I/LnJ oocytes underwentfertilization and cleavage to the 2-cell stage as oocytes fromanother inbred stain, C57BL/6J, and similar percentages of 2-cellstage oocytes completed the 2-cell stage to blastocyst transitionin vitro. However, unlike C57BL/6J oocytes, a much lower percentageof oocytes that matured in vivo in response to exogenous gonadotrophinsunderwent fertilization and cleavage to the 2-cell stage thanoocytes that underwent maturation in vitro. Likewise, lowerpercentages of 2-cell stage embryos derived from in-vivo maturedI/LnJ oocytes developed to blastocysts than embryos derivedfrom in-vitro matured oocytes. These results show than I/LnJoocytes are atypical in the progression of both nuclear andcytoplasmic maturation. These defects may account for the poorreproductive performance of I/LnJ mice. Thus, I/LnJ mice mightbe a useful model for studying infertility resulting from defectiveoocytes.     

Endocrinology: Efficacy of chronic therapy with the gonadotrophin releasing hormone agonist decapeptyl in patients with polycystic ovary syndrome

Our objective was to assess the endocrine and morphologicalresponse of polycystic ovary syndrome (PCOS) in patients receiving6 months of therapy with the long-acting gonadotrophin releasinghormone agonist (GnRH agonist) decapeptyl (3.75 mg monthly injections).Eighteen documented PCOS patients were basally evaluated forhirsutism, gonadotrophin and androgen concentrations and ovarianmorphology using trans-vaginal ultrasonography. Measurementswere repeated at 3 and 6 months. The results (values as x ±SD) showed a significant improvement in hirsutism (Ferrimanscore 11.0 ± 5.9 versus 6.6 ± 2.7, P < 0.01),acne and seborrhoea. A significant post-treatment decrease ingonadotrophins [follicle-stimulating hormone (FSH): 5.8 ±1.8 versus 3.8 ± 1.1 IU/I, P < 0.01; luteinizing hormone(LH): 10.8 ± 8.3 versus 3.4 ± 3.3 IU/1, P <0.01], LH/FSH ratio (1.8 ± 1.1 versus 0.8 ± 0.6,P < 0.01) and androgen concentrations (free testosterone:4.0 ± 1.9 versus 1.9 ± 0.7 pg/ml, P < 0.01,₄-androstenedione: 3.9 ± 1.2 versus 1.9 ± 0.6ng/ml, P < 0.001) was also found, while oestradiol approximatedcastration concentrations (68.4 ± 29.5 versus 29.1 ±6.7 pg/ml, P < 0.001). Finally, mean ovarian volume (19.7± 6.2 versus 10.9 ± 4.6 cm³, P < 0.001), capsulethickness (2.5 ± 0.8 versus 1.9 ± 0.7 mm, P <0.05) and stromal density dropped significantly, as did uterinevolume (34.2 ± 10.5 versus 19.9 ± 8.9 cm3, P <0.01). In conclusion, treatment of our PCOS patients for 6 monthswith the GnRH agonist decapeptyl proved efficient in inducingsignificant clinical, biochemical and ovarian morphologicalimprovement.     

Abnormal methylation of imprinted genes in human sperm is associated with oligozoospermia

Genomic imprinting marks in the male germ line are already establishedin the adult germinal stem cell population. We studied the methylationpatterns of H19 and MEST imprinted genes in sperm of controland oligozoospermic patients, by bisulphite genomic sequencing.We here report that 7 out of 15 (46.7%) patients with a spermcount below 10 x 10⁶/ml display defective methylation of H19and/or MEST imprinted genes. In these cases, hypomethylationwas observed in 5.54% (1.2–8.3%) and complete unmethylationin 2.95% (0–5.9%) of H19 clones. Similarly, for the CTCF-bindingsite 6, hypomethylation occurred in 4.8% (1.2–8.9%) andcomplete unmethylation in 3.7% (0–6.9%) of the clones.Conversely, hypermethylation occurred in 8.3% (3.8–12.2%)and complete methylation in 6.1% (3.8–7.6%) of MEST clones.Of the seven patients presenting imprinting errors, two hadboth H19 hypomethylation and MEST hypermethylation, whereasfive displayed only one imprinted gene affected. The frequencyof patients with MEST hypermethylation was highest in the severeoligozoospermia group (2/5 patients), whereas H19 hypomethylationwas more frequent in the moderate oligozoospermia (2/5 patients).In all cases, global sperm genome methylation analysis (LINE1transposon) suggested that defects were specific for imprintedgenes. These findings could contribute to an explanation ofthe cause of Silver–Russell syndrome in children bornwith H19 hypomethylation after assisted reproductive technologies(ART). Additionally, unmethylation of the CTCF-binding sitecould lead to inactivation of the paternal IGF2 gene, and belinked to decreased embryo quality and birth weight, often associatedwith ART.     

Back muscle as a promising site for ovarian tissue transplantation, an animal model

BACKGROUND: The aim of this study was to evaluate the optimal transplantationsite for ovarian tissue fragments in murine hosts. We comparedthe transplantation to the back muscle (B) versus the kidneycapsule (K) in a mouse allograft model. METHODS: Hemi-ovaries from 12-day-old mice were allografted into B andK of bilaterally ovariectomized same strain recipients whichhad undergone gonadotrophin stimulation (n = 15). Graft survivalafter 27 days, angiogenesis and follicle development were scoredand compared to age-matched control ovaries (38-day old, n =5). The ability of oocytes to be fertilized was studied afterIVF, ICSI and embryos were transferred to recipient mothers.Anti-mouse CD 31+ antibody was used to evaluate neo-vascularizationin grafts. RESULTS: Primordial follicle survival was higher (P < 0.01) and vascularsupport was better (P < 0.01) in B- than in K-grafts. From34 oocytes retrieved from B-grafts (15 metaphase I, of which14 matured in vitro, and 19 collected at metaphase II), 18 morulaewere obtained. Transfer of 12 embryos obtained by ICSI led tothree live offspring, and transfer of six IVF embryos to anotherrecipient mother yielded four offspring, one of which was borndead and one showed placental anomalies. CONCLUSIONS: The back muscle is a promising site for ovarian allografts inmice. This is the first report of live offspring obtained afterback muscle grafting using both IVF and ICSI.