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BACKGROUND: This study was designed to determine the efficacy of a programme using frozen donor semen in a cohort of patients returning for treatment after previously conceiving through donor insemination (DI). METHODS: The cohort included 222 couples with secondary infertility (previous live birth) in one University Hospital Centre. The treatment sequence involved DI cycles until completion. Live births, drop-out for personal or medical reasons and recourse to IVF with donor semen (IVF-D) were recorded for all patients. Live births were expressed as both rate per cycle and crude cumulative rate. RESULTS: At the end of the DI cycles, 65% of couples in the cohort achieved second parenthood. Most of them (77%) succeeded after only four DI cycles. The majority of couples who stopped treatment did so for personal reasons. CONCLUSIONS: Patients involved in a second parenthood project belong to a selected population. Management and counselling of such patients need to reconcile the early efficacy of DI cycles, the invasiveness of the IVF-D procedure and the availability of donor semen. [ABSTRACT FROM AUTHOR]

BACKGROUND Whether extended culture allowing selection of embryos with high development potential has any advantage over cleavage-stage embryo transfer remains a matter of debate. Among the currently unsolved questions, the cumulative delivery rate resulting from fresh and frozen embryo transfers needs to be taken into account in both strategies. The aim of our study was, therefore, to compare the efficacy of single embryo transfer either on Day 2 or on Day 5/6 combining fresh and frozen embryo transfers. METHODS A prospective study including 478 couples assigned on a voluntary basis to undergo elective single embryo transfer (eSET, n = 243) on Day 2 or single blastocyst transfer (SBT, n = 235) on Day 5/6 was performed. The primary outcome measurement was the cumulative delivery rate including fresh and frozen–thawed cycles in both groups. RESULTS The delivery rate per cycle following fresh embryo transfer was significantly higher in the SBT group compared with the eSET group (P CONCLUSIONS The observed cumulative delivery rates in this study do not allow us to take a position in favor of SBT or eSET. An improvement in blastocyst cryopreservation may change this attitude. [ABSTRACT FROM AUTHOR]

BACKGROUND Dialogue between the oocyte and cumulus cells is essential for oocyte maturation. A prospective laboratory research project was designed to evaluate transcription of specific genes in cumulus cells harvested before intracytoplasmic sperm injection from pre-ovulatory follicles, according to individual oocyte nuclear maturity and developmental competence. Genes were chosen because their expression was induced by the LH peak [Steroidogenic Acute Regulatory protein (STAR), Cyclooxygenase 2 (COX2 or PTGS2), Amphiregulin (AREG)] or because they were involved in oocyte lipidic metabolism [Stearoyl-Coenzyme A Desaturase 1 and 5 (SCD1 and SCD5)] or in gap-junctions [Connexin 43 (CX43 or GJA1)]. METHODS mRNA levels in cumulus cells were assessed by real-time PCR. RESULTS Expression levels of all genes investigated, except Cx43, were increased after resumption of meiosis. Nuclear maturation was thus associated with increased expression of STAR, COX2, AREG, SCD1 and SCD5 by cumulus cells. When considering only cumulus associated with metaphase II oocytes, gene expression was independent of morphological status at Day 2. In contrast, transcript levels were lower and distributed over a narrower range in cumulus enclosing oocytes achieving blastocyst development at Day 5/6 than in cumulus enclosing oocytes unable to develop beyond the embryo stage. CONCLUSION Further developmental potential from embryo to blastocyst stage was associated with lower expression in a narrow range for these genes. [ABSTRACT FROM AUTHOR]

: BACKGROUND Non-invasive and routine developmental markers are available to select the most viable embryo; however their respective values in terms of blastocyst development potential remain difficult to distinguish. : METHODS During this prospective study, the sequential growth of 4042 embryos individually cultured from day 1 to day 5/6 was recorded. Pronuclear morphology on day 1, and early cleavage, cell number and fragmentation rate on day 2 were evaluated for each zygote. Additionally, blastocyst transfers were analysed with regard to their implantation ability and early embryo development parameters. : RESULTS Once adjusted to each other, each of the four parameters remained related to blastocyst development. Early cleavage and cell number on day 2 were the most powerful parameters to predict the development of a good morphology blastocyst at day 5. Moreover, whereas transfers of a good morphology blastocyst were associated with high implantation and live birth rates, parameters of early development were not helpful in predicting their implantation ability. : CONCLUSIONS The combination of all four parameters allowed the prediction of blastocyst development with an area under the receiver operating characteristics curve of 0.688, which represents a fairly low prediction of embryo viability. Such results indicate that it is necessary to search for additional criteria, including the ability of the blastocyst to develop. [ABSTRACT FROM AUTHOR]

Background: The ability to predict the developmental and implantation ability of embryos remains a major goal in human assisted-reproductive technology (ART) and most ART laboratories use morphological criteria to evaluate the oocyte competence despite the poor predictive value of this analysis. Transcriptomic and proteomic approaches on somatic cells surrounding the oocyte (granulosa cells, cumulus cells [CCs]) have been proposed for the identification of biomarkers of oocyte competence. We propose to use a Reverse Phase Protein Array (RPPA) approach to investigate new potential biomarkers of oocyte competence in human CCs at the protein level, an approach that is already used in cancer research to identify biomarkers in clinical diagnostics., Methods: Antibodies targeting proteins of interest were validated for their utilisation in RPPA by measuring siRNA-mediated knockdown efficiency in HEK293 cells in parallel with Western blotting (WB) and RPPA from the same lysates. The proteins of interests were measured by RPPA across 13 individual human CCs from four patients undergoing intracytoplasmic sperm injection procedure., Results: The knockdown efficiency of VCL, RGS2 and SRC were measured in HEK293 cells by WB and by RPPA and were acceptable for VCL and SRC proteins. The antibodies targeting these proteins were used for their detection in human CCs by RPPA. The detection of protein VCL, SRC and ERK2 (by using an antibody already validated for RPPA) was then carried out on individual CCs and signals were detected for each individual sample. After normalisation by VCL, we showed that the level of expression of ERK2 was almost the same across the 13 individual CCs while the level of expression of SRC was different between the 13 individual CCs of the four patients and between the CCs from one individual patient., Conclusions: The exquisite sensitivity of RPPA allowed detection of specific proteins in individual CCs. Although the validation of antibodies for RPPA is labour intensive, RRPA is a sensitive and quantitative technique allowing the detection of specific proteins from very small quantities of biological samples. RPPA may be of great interest in clinical diagnostics to predict the oocyte competence prior to transfer of the embryo using robust protein biomarkers expressed by CCs.

The introduction of intracytoplasmic sperm injection (ICSI) provided an effective treatment for infertile couples whose infertility was attributed to male factors. However, some of them face poor results after ICSI and subsequently use artificial insemination with donor sperm (AID). Only a few studies have reported on the clinical outcome of AID cycles after previous failed ICSI cycles, with contrasting results. The results reported here involve a cohort of 47 couples undertaking 175 AID cycles after 120 failed ICSI cycles for various reasons. Couples were allocated to two groups according to the availability of top quality embryos (TQE) in ICSI cycles. In our series, AID was successful for couples with and without TQE previously transferred in ICSI cycles, the live birth rate (LBR) per cycle being 20.0% and 13.3%, respectively. However, couples with TQE tended to succeed more rapidly than couples with poor quality embryos, with a higher cumulative LBR (68.0% versus 54.5%, respectively). These findings demonstrate that even couples with a history of unsuccessful ICSI cycles because of poor embryo quality are able to achieve high LBR after AID cycles. However, such couples have a lower cumulative LBR and are required to be more patient to achieve parenthood., (© 2012 Blackwell Verlag GmbH.)

Object: The prediction of embryo viability by usual morphological analysis is currently unsatisfactory. New non-invasive techniques such as high-resolution nuclear magnetic resonance ((1)H-NMR) spectroscopy that allows assessment of metabolic profiling in spent culture media might help embryologists to predict embryo development., Materials and Methods: Individual microdrops of culture media were analysed after 24 h of embryo culture (from day 3 to day 4) by spectroscopy using a 1 mm microliter probe allowing analysis without sample dilution. Embryos were divided into two groups on day 5: non-arrested embryos (n = 19) and arrested embryos unable to reach the blastocyst stage (n = 20). Multivariate analysis techniques such as Principal Component Analysis (PCA) and Orthogonal Partial Least Square Discriminant Analysis (OPLS-DA) were performed to compare extracellular metabolite balance., Results: (1)H-NMR used in combination with a 1 mm probe suggested that in vitro cultured human embryos that have a high developmental potential modify their environment slightly compared to embryos that cease to develop. However, differences between the two groups did not reach statistical significance and multivariate statistical analysis did not allow clustering of the two groups., Conclusion: This study indicated that this technique would not be sufficiently powerful alone to provide information that might help to assess the developmental potential of individual embryos for in vitro fertilisation (IVF).

Study Question: Does BCAR4 have a role in mammalian embryo development?, Summary Answer: Expression, localization and functional data support that BCAR4 is a maternal-effect protein in non-rodent mammals., What Is Known Already: BCAR4 was previously identified as an oocyte-specific gene in cattle, and as a marker of certain breast tumors in humans., Study Design, Size, Duration: Human oocytes were obtained from patients undergoing IVF, but had failed to mature after ovarian stimulation. Dog oocytes were obtained from ovariectomized bitches. Pig, horse and bovine ovaries were obtained from commercial slaughterhouses for extraction of immature oocyte-cumulus complexes. In vivo matured bovine matured oocytes were obtained after ovulation induction and ovulation inducing treatment of Montbeliard heifers., Materials, Setting and Methods: Expression at the RNA level was analyzed by reverse transcription coupled to polymerase chain reaction. Western blot and immunolabeling coupled to confocal or electronic microscopy were used to analyze bovine protein expression and intracellular localization. For the functional approach, short-interfering RNA were microinjected into mature bovine oocytes, followed by IVF; cleavage and embryo development were recorded., Main Results and the Role of Chance: The BCAR4 gene is conserved in mammalian species from various orders and has been lost in rodents after divergence with lagomorphs. The transcript is expressed in the oocytes of humans and domestic species. We bring the first experimental evidence of the BCAR4 protein in mammals. In cattle, the protein is not detected in immature oocytes but starts to be synthesized during maturation, increases in the zygote and persists until the morula stage. The protein is detected throughout the cytoplasm in mature oocytes, concentrates in and around the pronuclei in the zygote, and appears to shuttle in and out of the nuclei starting in the 2-cell embryo; BCAR4 is also present at the junctions between blastomeres from 2-cell to morula. In our functional approach, targeting the BCAR4 transcript by small-interfering RNA significantly compromised development to the morula or/and blastocyst stages (P < 0.05, logistic regression)., Limitations, Reasons for Caution: As indicated above, protein expression and function were investigated in cattle and mostly in vitro matured oocytes were used., Wider Implications of the Findings: This study provides a novel candidate gene whose mutation or deregulation may underlie certain cases of unexplained female infertility.

Background: Single embryo transfer (SET) is the most successful way to reduce the frequency of multiple pregnancies following in vitro fertilisation. However, selecting the embryo for SET with the highest chances of pregnancy remains a difficult challenge since morphological and kinetics criteria provide poor prediction of both developmental and implantation ability. Partly through the expression of specific genes, the oocyte-cumulus interaction helps the oocyte to acquire its developmental competence. Our aim was therefore to identify at the level of cumulus cells (CCs) genes related to oocyte developmental competence., Methodology/principal Findings: 197 individual CCs were collected from 106 patients undergoing an intra-cytoplasmic sperm injection procedure. Gene expression of CCs was studied using microarray according to the nuclear maturity of the oocyte (immature vs. mature oocyte) and to the developmental competence of the oocyte (ability to reach the blastocyst stage after fertilisation). Microarray study was followed by a meta-analysis of the behaviour of these genes in other datasets available in Gene Expression Omnibus which showed the consistency of this list of genes. Finally, 8 genes were selected according to oocyte developmental competence from the 308 differentially expressed genes (p<0.0001) for further validation by quantitative PCR (qPCR). Three of these 8 selected genes were validated as potential biomarkers (PLIN2, RGS2 and ANG). Experimental factors such as inter-patient and qPCR series variability were then assessed using the Generalised Linear Mixed Model procedure, and only the expression level of RGS2 was confirmed to be related to oocyte developmental competence. The link between biomarkers and pregnancy was finally evaluated and level of RGS2 expression was also correlated with clinical pregnancy., Conclusion/significance: RGS2, known as a regulator of G protein signalling, was the only gene among our 8 selected candidates biomarkers of oocyte competence to cover many factors of variability, including inter-patient factors and experimental conditions.

BACKGROUND Embryologists currently face a challenge when counselling patients regarding the stage and the number of embryos to transfer when no top-quality embryos (TQE) are available. METHODS The aim of this study was to evaluate the efficacy of single blastocyst transfer (SBT) in comparison with the transfer of two cleavage-stage embryos in women under 36 years old. A total of 450 women under 36 years undergoing their first or second IVF treatment who had no TQE on Day 2 were included in this prospective study. Couples were assigned to either a SBT or a double cleavage-stage embryo transfer (DET). The clinical end-points monitored were rates of implantation, delivery and multiple deliveries. RESULTS The rate of transfer was significantly lower for couples assigned to the SBT group compared with the DET group (88 versus 100%, respectively, P < 0.001) while the delivery rate per oocyte retrieval was similar in both groups (26.7%). By contrast, the rate of multiple deliveries was significantly lower in the SBT group compared with the DET group (3.3 versus 23.3%, respectively, P < 0.01). Blastocyst cryopreservation was twice as high in the SBT group compared with the DET group (39 versus 18%, respectively, P < 0.001). CONCLUSIONS These findings show the value of extended embryo culture for couples without TQE. In such situations, delaying embryo transfer in order to select a single blastocyst with the highest potential for implantation can reduce the number of multiple pregnancies. Furthermore, our results demonstrate that extended culture allows blastocyst cryopreservation from embryos not available for Day 2 cryopreservation.

Routine early developmental parameters are widely used in IVF centres to evaluate embryo development and fresh single-blastocyst transfer currently seems superior to single-embryo transfer. Would early morphological parameters help to choose the single blastocyst to be transferred, thereby improving the chances of implantation and live birth rate? This prospective observational study analysed the individual outcomes of 2617 embryos from 511 IVF couples scheduled for a single-blastocyst transfer. Embryo and blastocyst scores were constructed. There was a clear relationship between the kinetics and morphology of blastocysts and further implantation and live birth rate. There was a limited predictive value of embryo score with regard to blastocyst development and growth kinetics. Implanted and non-implanted blastocysts showed similar embryo scores. Thus usual morphological parameters on days 1 and 2 seem to have no additional value in indicating the right blastocyst to transfer. Non-invasive approaches might be helpful to increase the chances of implantation in the future., (Copyright © 2010 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

Objective: To describe a case of conjoined twins after intracytoplasmic sperm injection (ICSI) and transfer of a single day 2 embryo., Design: Case report., Setting: University hospital., Patient(s): A 30-year-old woman underwent ICSI with a transfer of a single day 2 embryo., Intervention(s): Vaginal ultrasound examination was performed during early pregnancy., Result(s): The diagnosis of conjoined twins was made by vaginal ultrasonography at 9 weeks' gestation. The conjoined fetuses were diagnosed as thoracopagus twins with a single beating heart shared between the thoraxes. Extensive ultrasound examination suggested an extremely poor prognosis, Therapeutic termination was performed at 11 weeks., Conclusion(s): Given that single embryo transfers are able to shed light on the real incidence of conjoined twins developed on day 2/3 or on day 5/6, this is of interest mechanistically. Thus, it should be taken into account when trying to determine the true incidence of conjoined twins and ultimately trying to understand why these anomalies occur. The importance of early expert vaginal ultrasonography in pregnancies resulting from assisted reproductive technologies is emphasized., (Copyright 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Purpose: While extended culture has been considerably improved, some questions remain regarding the application of Single Blastocyst Transfer (SBT)., Methods: An observational cohort study was undertaken with 456 women under 36 years old and assigned to SBT on a voluntary basis. The main outcome was the cumulative delivery rate per couple according to the number of Top Quality Embryos (TQE) on day 2 (Group 1= > or =2 TQE, Group 2= 1 TQE and Group 3= 0 TQE)., Results: Rate of transfer and mean number of frozen blastocyts were higher in Group 1 compared to Group 3. As a consequence, the cumulative delivery rate per couple was higher in Group 1 (47.9%) compared to Group 3 (34.9%)., Conclusions: Single blastocyst transfer combining fresh and frozen cycles, might be a worthwhile strategy irrespective of embryo quality on day 2 providing good delivery rates while keeping the rate of multiple deliveries low.

The 'ESHRE Guidelines for Good Practice in IVF Laboratories' were drawn up by the Special Interest Group (SIG) in Embryology and published in the year 2000, and since then they constitute the minimal requirements for any laboratory offering assisted reproduction techniques (ART). In the understanding that the embryologist has a responsibility for the correct and justified application of ART in the laboratory, the implementation of these guidelines requires a quality management programme to be in place that encompasses and integrates the operative units, the processes and procedures that represent the core of ART clinics. In March 2004, the European Parliament issued the Directive 2004/23/EC 'On setting standards of quality and safety for the donation, procurement, testing, processing, preservation, storage and distribution of human tissues and cells'. The Directive applies to human tissues and cells, including fresh or frozen reproductive cells for application to the human body, and is mainly concerned with increasing quality and safety through the implementation of a quality management system. Therefore, the European Society of Human Reproduction and Embryology (ESHRE) undertook a series of initiatives aiming to promote assurance of good laboratory practice and to define the concept of qualified embryologists. One ESHRE initiative was to revise the guidelines for good practice in IVF laboratories, not only in response to the need of embryologists for support and guidance in their duties, but also as a complement to the requirements issued by the Tissue and Cell Directive. The SIG in Embryology hopes that this document may assist the laboratory staff to operate according to the requirements of harmonization, implementation, inspection and certification that are now common to all European member states.

Background: Zygote arrest 1 (ZAR1) is one of the few known oocyte-specific maternal-effect genes essential for the beginning of embryo development discovered in mice. This gene is evolutionary conserved in vertebrates and ZAR1 protein is characterized by the presence of atypical plant homeobox zing finger domain, suggesting its role in transcription regulation. This work was aimed at the study of this gene, which could be one of the key regulators of successful preimplantation development of domestic animals, in pig and cattle, as compared with human., Methods: Screenings of somatic cell hybrid panels and in silico research were performed to characterize ZAR1 chromosome localization and sequences. Rapid amplification of cDNA ends was used to obtain full-length cDNAs. Spatio-temporal mRNA expression patterns were studied using Northern blot, reverse transcription coupled to polymerase chain reaction and in situ hybridization., Results: We demonstrated that ZAR1 is a single copy gene, positioned on chromosome 8 in pig and 6 in cattle, and several variants of correspondent cDNA were cloned from oocytes. Sequence analysis of ZAR1 cDNAs evidenced numerous short inverted repeats within the coding sequences and putative Pumilio-binding and embryo-deadenylation elements within the 3'-untranslated regions, indicating the potential regulation ways. We showed that ZAR1 expressed exclusively in oocytes in pig ovary, persisted during first cleavages in embryos developed in vivo and declined sharply in morulae and blastocysts. ZAR1 mRNA was also detected in testis, and, at lower level, in hypothalamus and pituitary in both species. For the first time, ZAR1 was localized in testicular germ cells, notably in round spermatids. In addition, in pig, cattle and human only shorter ZAR1 transcript variants resulting from alternative splicing were found in testis as compared to oocyte., Conclusion: Our data suggest that in addition to its role in early embryo development highlighted by expression pattern of full-length transcript in oocytes and early embryos, ZAR1 could also be implicated in the regulation of meiosis and post meiotic differentiation of male and female germ cells through expression of shorter splicing variants. Species conservation of ZAR1 expression and regulation underlines the central role of this gene in early reproductive processes.

Clinicians who treat unsuccessful couples despite repeated transfers of good quality embryos face a challenge. Among the various strategies that have been described, embryo transfer at the blastocyst stage has been postulated to improve implantation. A prospective non-randomized analysis was performed in 276 IVF patients who failed to conceive after at least two early embryo transfers of at least two grade 1-2 embryos per cycle. For the next attempt, couples chose between day 2 embryo transfer (D2 group; n = 147) and day 5/6 blastocyst transfer (D5/D6 group; n = 129) before starting the following attempt. Embryo quality was assessed and results were expressed as clinical pregnancy, live birth and implantation rates per cycle. Embryo grade 1 number was similar between both groups, whereas mean embryo score of the whole cohort was slightly higher in the D2 group. The live birth rates per cycle (27.9 versus 19.7%) and implantation rates per cycle (25.4 versus 12.4%) were higher in the D5/D6 group compared with the D2 group. Improved embryo selection and uterine receptivity may explain the additional benefit of embryo transfer at the blastocyst stage for couples with repeated implantation failures.

A cohort follow-up study was designed to compare the efficacy of IVF using frozen donor semen (IVF-D) following previously failed DI cycles (unexplained female infertility) and direct IVF-D treatment because of tubal infertility (control group). The cohort comprised 189 couples initiating IVF-D after previously failed DI cycles (n = 126) or directly (n = 63). Couples were followed until completion (success or drop-out for personal or medical reasons). Live births and drop-out were expressed both as rate per cycle and crude cumulative rate. Characteristics of IVF-D cycles were similar between the two groups. Moreover, overall outcome was also similar in terms of crude cumulative live birth rate (54.0 versus 57.1% for failed DI cycles and tubal infertility groups respectively). This is the first report on crude cumulative live birth rate based on a cohort follow-up study in unexplained previously failed DI cycles and tubal infertility. Previously failed DI cycles did not impair the chances of achieving a successful pregnancy using IVF-D in this series. Slight oocyte dysfunction, which might underlie the failure of DI cycles, might be overcome using IVF-D.

A study was undertaken to determine whether the interval between thawing and transfer influences both biological and clinical outcomes of cryopreserved blastocysts, using supernumerary embryos cultured in sequential media. One hundred and seventy-two patients who underwent blastocyst thawing without any exclusion criteria were included in this single center prospective study of blastocyst thawing cycles. Outcome of 338 blastocysts originating from culture of supernumerary embryos in sequential media was analyzed after 4 or 20 h of culture between thawing and transfer. Survival rate, re-expansion and hatching rates for surviving blastocysts, implantation rates (IRs), pregnancy and miscarriage rates were studied. Blastocyst survival was not influenced by the incubation time after thawing; however both re-expansion and hatching rates were increased after 20-h incubation. Moreover, the IR per thawed or transferred blastocyst was increased three-fold after 20-h incubation compared to 4-h incubation. Increasing the interval between thawing and transfer appears to be beneficial in order to better select for transfer frozen-thawed blastocysts.

The role of the proto-oncogene Kit expression during gonadal development, then in differentiated spermatogonia has been thoroughly established. The present study was designed to investigate the consequences of a partial defect in Kit gene expression on sperm fertilizing ability, using Kit haplodeficient mice (kitW-lacZ/+). Same inbred mice (kit+/+) were used as controls. Epididymal sperm characteristics and in vivo fertility were assessed, then in vitro-fertilization experiments were carried out for mice of both genotypes. Epididymal sperm count was drastically reduced, and sperm motility was also decreased in kitW-lacZ/+ compared with kit+/+ males. Both in vivo or in vitro fertility were greatly reduced in kitW-lacZ/+ compared with kit+/+ males. By contrast, the fertility of kitW-lacZ/+ females was apparently unaffected. Additionally, a higher number of spermatozoa with undetected acrosomal contents was revealed by fluorescein isothiocyanate-labelled Pisum sativum agglutinin acrosomal staining after epididymal sperm retrieval in kitW-lacZ/+ mice, whereas no difference was observed after induction of acrosomal reaction in mice of either genotype. Ultra-structural data confirmed the higher frequency of abnormal acrosome in spermatozoa of kitW-lacZ/+ mice. Thus, sperm production is impaired in Kit haplodeficient mice both on a quantitative and a qualitative basis. Finally, we show that one single copy of Kit gene is not sufficient to maintain genuine fertility in male mice.

Kit/stem cell factor (SCF ) has been reported to be involved in survival and proliferation of male differentiating spermatogonial cells. This kinetics study was designed to assess the role of Kit/SCF during spermatogenesis in mice, and the extent of male programmed germ cell death was measured between 8 and 150 days of age. For this purpose, 129/Sv inbred mice in which one Kit allele was inactivated by an nlslacZ sequence insertion (Kit(W-lacZ/+)) were compared with 129/Sv inbred mice with wild-type alleles at the Kit locus. Four different approaches were used: 1) morphometric study to assess spermatogenesis, 2) flow cytometry to study testicular cell ploidy, 3) in situ end labeling to detect apoptosis, and 4) follow-up of reporter gene expression. Spermatogenesis was lower in Kit(W-lacZ/+) heterozygous mice both at the onset of spermatogenesis and during adulthood. Indeed, greater apoptosis occurred at the onset of spermatogenesis. This was followed in the adult by a smaller seminiferous tubule diameter and a lower ratio between type B spermatogonia and type A stem spermatogonia in Kit(W-lacZ/+) mice compared with Kit(+/+) mice. These differences are probably related to the Kit haplodeficiency, which was the only difference between the two genotypes. Germ cell counts and testicular cell ploidy revealed delayed meiosis in Kit(W-lacZ/+) mice. Reporter gene expression confirmed expression of the Kit gene at the spermatogonial stage and also revealed Kit expression during the late pachytene/diplotene transition. These results suggest involvement of Kit/SCF at different stages of spermatogenesis.

Background: This study was designed to determine the crude cumulative live-birth rates in a cohort initiating frozen donor semen treatment until completion., Methods: This cohort study included 588 couples with primary infertility in one University Hospital centre. The treatment sequence involved first artificial insemination (AID) followed by IVF if necessary (IVF-D). Live birth, drop-out for personal or medical reasons and recourse to IVF-D were recorded for all patients. Live births and drop-out were expressed both as rates per cycle and crude cumulative rates., Results: At the completion of AID and IVF-D cycles, 406 couples in the cohort (69%) achieved a live-birth and 182 couples (31%) discontinued treatment. In most cases, couples stopped treatment for personal reasons (74%) whereas fewer couples were denied further treatment for medical reasons (26%)., Conclusions: This is the first report on the crude cumulative live-birth rate in a cohort after AID and IVF-D cycles. Although calculation based on crude cumulative live-birth rate shows lower results in comparison with life table analysis, this method allows patients to obtain an insight into their actual chances of achieving a successful pregnancy.

Background: The purpose of this study was to evaluate the respective influences of blastomere survival and resumption of mitosis on the outcome of frozen-thawed embryos., Methods: A retrospective analysis was performed in our centre on 363 thawing cycles, involving 4-cell day 2 grade 1 embryos with <10% fragmentation., Results: A higher implantation rate per transferred embryo was observed when all transferred embryos were characterized by fully intact blastomeres (100% blastomere survival) as compared with damaged embryos (50 or 75% blastomere survival) (22.0 versus 7.2%; P < 0.0001). Moreover, the implantation rate per transferred embryo was significantly higher for cleaved embryos compared with uncleaved embryos (19.7 versus 3%; P < 0.0001). Transfer of fully intact, cleaved embryos resulted in the highest implantation rates compared with transfer of damaged and uncleaved embryos (27.4 versus 0%; P < 0.0001). Intermediate implantation rates were observed when only one of the two criteria was fulfilled (13 versus 11% respectively; P > 0.05). Multivariate analysis showed that the clinical pregnancy rate was influenced by both criteria (odds ratio = 3.4 for transfer of embryos with six or more cells versus embryos with less than six cells., Conclusion: The results of our study suggest that the most important factor to predict further embryo development is the total number of blastomeres in transferred embryos, however they are obtained (good survival and/or resumption of mitosis).

The need to assess the health of children born after assisted reproductive technologies (ART) using frozen donor spermatozoa has been a major concern for the last decade. Most available published studies are confusing (since they are retrospective) and refer to small numbers of cases or involve a bias. Two prospective and large population-based studies have been published, one in France and the other in Australia. The general characteristics of children born after ART using frozen spermatozoa was unchanged in comparison with the general population (weight, prematurity, stillbirths, sex ratio). However, the rate of multiple pregnancy was increased, first in artificial insemination with donor semen (AID), where the use of hormonal treatment may be too heavily prescribed, and second in IVF with donor semen (IVF/D) where both the ovulation induction treatments and embryo transfer policy increase the rate of multiple pregnancies. As far as birth defects or chromosomal abnormalities are concerned, no difference has been observed from the general population. Finally, the use of frozen spermatozoa does not seem to affect the health of children conceived by AID or IVF/D. The psychosocial development of such infants is not as well known because of the confidentiality in many countries concerning sperm donation. However, the available information on the psychosocial development of these children up to the age of 8-10 years appears to be reassuring. Larger and longer studies are needed to answer such questions, particularly with regard to adulthood.

Objective: To determine whether hMG offers an advantage over clomiphene citrate (CC) in achieving pregnancy after IUI with husband's sperm., Design: Randomized prospective trial., Setting: Infertility patients in a university teaching hospital., Patient(s): Fifty-eight women under 39 years old undergoing ovulation induction before IUI., Intervention(s): The women were assigned randomly to one of two treatment groups. Patients in group I (CCHH) received CC for the first two cycles and hMG for the last two cycles. Patients in group II (HHCC) received hMG for the first two cycles and CC for the last two cycles., Main Outcome Measure(s): Cycle fecundity rates for the two treatment modalities were compared statistically with use of life-table analysis., Result(s): Of the 174 cycles studied, overall cycle fecundity rate was 11.11 (9 of 81 cycles) in the CCHH group and 10.75 (10 of 93 cycles) in the HHCC group. The difference was not statistically significant. The cycle fecundity rate was 14.44% (13 of 90 cycles) for cycles with CC and 7.14% (6 of 84) with hMG. The difference was not statistically significant., Conclusion(s): These data suggest that CC is an effective alternative to hMG in the population examined.

Both the ability to freeze human spermatozoa and the possibility of pregnancy following intrauterine insemination have existed for >40 years. There have been a number of improvements during that time concerning the methods of freezing and thawing human spermatozoa. Initially, the use of the cryoprotective properties of glycerol allowed a major improvement; subsequently, changes were mainly empirical. It was a long time before specific cryobiological studies were undertaken. However, the necessity for these became apparent with the partial recovery or sometimes loss of motility after freezing either subfertile semen before chemotherapy or radiotherapy, or spermatozoa collected from non-physiological situations (epididymal or testicular spermatozoa). The main trends in improvement have defined end-points other than the percentage of motility recovery or the assessment of ultrastructural damage. More sensitive criteria of the objective assessment of motility, energy status, damage to the plasma membrane or to subcellular elements, chromatin stability and chromosomal damage have been proposed as complementary end-points to better assess sperm cryopreservation. A different approach was related to the biochemical environment and physical conditions imposed on spermatozoa during the freezing and thawing process. Biochemical changes were assessed following different combinations of various extenders which attempted either to better preserve some parameter or to avoid the tendency towards drastic increase in osmotic pressure. Analysis of physical conditions was linked to the rate of cooling, freezing and warming, and was based on cryobiological studies. Finally, even though such improvements are not negligible, many questions remain unanswered. The extensive use of frozen spermatozoa during assisted reproductive techniques, together with the development of assisted fertilization using surgically collected spermatozoa, creates the need for additional studies to improve the cryopreservation of human spermatozoa.

Purpose: Our purpose was to determine the obstructed epididymal epithelium morphometric state and to elucidate the influence of morphometric parameters on the fertilizing ability of aspirated spermatozoa., Methods: During MESA with IVF, epididymal tissue samples were carefully dissected, fixed, and prepared for morphometric analysis. The epithelium of the three regions was studied: head, body, and tail (when present). The total area/luminal area ratio, epithelium thickness, and stereocilium length were measured., Results: We have found significant differences in the total area/luminal area ratio, epithelium thickness, and stereocilium length in obstructed epithelium length > 4 versus 0.5-2 cm (P < or = 0.01). Epithelium thickness and stereocilium length were significantly lower when no fertilization was observed in IVF., Conclusions: These results suggest that morphometric data of obstructed epididymis epithelium can be used as a means of explaining the failure of IVF with epididymal spermatozoa.

The internal pH (pHi) of human spermatozoa was measured by the fluorescent indicator 2,7-bicarboxyethyl-5,6-carboxyfluorescein acetoxymethyl ester (BCECF-AM) and the distribution of the radioactive [14C]-methylamine under different external ionic conditions. The effect of the addition of progesterone and human follicular fluid (HFF) on the spermatozoa pHi was also analysed. The pHi values obtained were almost identical with the two probes used. In sodium (NaM) and potassium (KM) media, a linear relationship between the internal and external pH was observed. In NaM, the pHi values were approximately 0.4 pH unit less than the external pH. In KM, the pHi measured was higher than in NaM and only slightly inferior to the external pH (0.1-0.2 pH unit). Addition of 10 microM progesterone, oestradiol 17 beta or 20% HFF to spermatozoa incubated at pH 7.2 in NaM did not induce any rapid variation of the BCECF fluorescence or change in the accumulation of methylamine. A slight change in pH (approximately 0.5 units) occurred with progesterone after 15 min. As a control, addition of 10 mM of NH4Cl induced a rapid alkalinization (0.4 pH unit) of the cell interior while 10 mM lactate produced only a slight acidification (approximately 0.2 pH unit). Under the same conditions (NaM, pH 7.2), the pHi of the spermatozoa prepared by Percoll gradient was found more acidic by 0.2 pH unit than washed unfractionated spermatozoa. Progesterone, oestradiol 17 beta and HFF had no effect on the pHi of these spermatozoa. The results obtained in this study show that it is possible to measure accurately the internal pH of human spermatozoa. Internal pH was found to be dependent upon the pH of the external medium and a quasi-linear relationship exists between the internal and external pH, suggesting no specific pH regulatory mechanisms. Our data suggest instead that the protons, under our experimental conditions, are passively distributed. Progesterone, oestradiol 17 beta and HFF, known to promote both capacitation and the acrosome reaction, do not act through a rapid pHi change.

This study measured glycosaminoglycans (GAGs) in human follicular fluid (hFF) obtained from patients undergoing hormonal stimulation with combined GnRH agonist followed by gonadotropin hormone (hMG or FSH) for in vitro fertilization (IVF). The GAGs were partially characterized through the use of various mucopolysaccharidases and then their effects tested on human sperm motility. In hFF, the GAG and protein concentrations were 4.4 +/- 1.3 mg/L and 32.6 +/- 3.2 g/L respectively. Chondroitins (CS) and hyaluronic acid (HA) significantly stimulate sperm motion in comparison to the control. The oocytes inseminated with GAGs-pretreated spermatozoa showed a significantly higher rate of cleavage and pregnancy or delivered number. The present results suggest that hFF is a rich source of sulfated glycosaminoglycan (CS), and non-sulfated glycosaminoglycan (HA). The stimulatory effects of hFF on human sperm motility may well depend on CS and HA. GAGs pretreated sperm may enhance the fertilizing ability of spermatozoa and thus the IVF outcome.

The effect of human follicular fluid (hFF) on the cholesterol and phospholipid content and the movement characteristics of human spermatozoa were studied. Semen was selected by a discontinuous Percoll gradient and incubated during in vitro capacitating conditions with B2 medium supplemented with hFF 20%. Percoll pelleted spermatozoa were incubated in either B2 (B2-Percoll) or B2 supplemented with hFF (hFF-Percoll). In hFF-Percoll, we observed a time-dependent (24 h) decrease in both the cholesterol and phospholipid contents (cholesterol: 10.1 vs. 8.7 nmol 10(-7) spermatozoa; phospholipids: 17.5 vs. 15.7 nmol 10(-7) spermatozoa, P < 0.05). This decrease in cholesterol and phospholipids in human spermatozoa was concomitant with a high straight line velocity, a high progressive motility percentage and an increased value of lateral head displacement without any significant alteration of the spermatozoal membrane. No modification of the cholesterol: phospholipid ratio after 2 and 24 h of incubation in either B2-Percoll (0.61, 0.54) in hFF-Percoll (0.59, 0.63) was observed when compared with original control semen. It is suggested that the decrease in cholesterol and phospholipids in hFF-Percoll may be taken into account for the changes of membrane modification as part of the capacitation process.

The purpose of this study was to investigate whether the measurement by NMR of some biochemical markers such as glycerylphosphorylcholine (GPC), glycerylphosphorylethanolamine (GPE), citrate, and lactate in human seminal plasma may be able to differentiate spermatogenic failure from obstructive azoospermia. The peak area ratios citrate/lactate, between normozoospermic and spermatogenic failure or obstructive azoospermia groups were significantly different. We have also found significant differences in GPE/GPC peak intensity ratio between spermatogenic failure men and obstructive azoospermia patients (P < 0.001). This ratio appears to be a very important parameter to differentiate spermatogenic failure from obstructive azoospermia. These results demonstrate the potential use of 1H NMR on human seminal plasma in male infertility evaluation and such analysis may be used to elucidate the molecular basis of the human pathologic seminal plasma disturbances.

A case report of delayed delivery of a quadruplet pregnancy is presented. This quadruplet pregnancy resulted from in-vitro fertilization. To our knowledge, this case represents the first report of quadruplets delivered on three separate days using the technique of delayed interval delivery. All infants survived and are healthy 2 years later.

Glycerylphosphorylcholine (GPC), glycerylphosphorylethanolamine (GPE), citrate, and lactate content of human seminal plasma was analysed by measuring the peak area of 1H nuclear magnetic resonance (NMR) spectra in samples from four groups of patients: 21 spermatogenic failure subjects; 14 obstructive azoospermic subjects (vasectomized); seven patients presenting very severe oligoasthenozoospermia (OAT) and 18 normozoospermic subjects (control). The peak areas for GPC, citrate and lactate in seminal plasma were significantly smaller for patients with azoospermia than for the control group: 16.79, 8.18 and 2.28 versus 23.38, 10.58 and 4.30, respectively (P < 0.01). The peak area ratios for citrate:lactate and GPC:lactate were significantly different (P < 0.01) between the control group and the spermatogenic failure or obstructive azoospermia groups. A significant difference was also found in GPE:GPC peak intensity ratio between spermatogenic failure and obstructive azoospermia subjects (P < 0.001). These results provide some quantitative markers which may be used for examining infertility by using 1H NMR of seminal plasma samples.

In order to evaluate the role of human follicular fluid (HFF) on the fertilizing capacity of spermatozoa, we studied the effect of HFF on the lipid composition and on the movement characteristics of human spermatozoa. Spermatozoa (spz) from normospermic patients were prepared with a discontinuous Percoll gradient and incubated in Ménézo B2 medium with or without a supplement of 20% HFF (HFF-Percoll spz and B2-Percoll spz respectively) for 2 and 24 h. After 2 h HFF incubation, percentage progressive motility, straight line velocity (VSL), and amplitude of lateral head displacement (ALH) were improved in HFF-Percoll spz as compared to B2-Percoll spz (P < or = 0.05). After a longer incubation period (24 h), lipid changes appeared in HFF-Percoll spz with lower levels of cholesterol (P = 0.02) and phospholipids (P = 0.05). No modification of the cholesterol/phospholipid ratio after 2 and 24 h of incubation in either B2-Percoll spz or HFF-Percoll spz was observed. Such decreases in lipid content of HFF-Percoll spz may be factors which could be taken into account as constituting part of membrane modifications during the capacitation process.

An original, nonradiometric method has been developed for studying the binding parameters of native follicle-stimulating hormone (FSH) to its specific receptors in human ovarian granulosa cells. After binding and washing of the cells, hFSH was desorbed from its receptors and quantitatively measured by a specific enzyme immunoassay (EIA) in which nonspecific binding was estimated in the presence of an excess of equine chorionic gonadotropin (eCG/PMSG), which binds to human FSH receptors but does not interfere in the hFSH EIA. This method makes use of native nonmodified hFSH molecules (in contrast to radiometric methods) and permits direct estimation of the binding parameters (Kd and total number of sites). The Kd of hFSH for its human granulosa receptors measured by this technique (4.8 +/- 0.3 x 10(-10) M) is close to that determined by other methods. However, we found a total number of specific FSH receptors per granulosa cell (1 to 6 x 10(4) higher than that reported by others by Scatchard analysis of competition dose-response curves in radioreceptor assays. The method is also sensitive enough to measure the in vivo occupancy of receptors by endogenous hFSH, which was found to be less than 6% in women undergoing hormonal treatment for in vitro fertilization.

A previous cytochemical study revealed that chromatin alterations could be induced in frozen-thawed human sperm. In order to evaluate the biological consequences of such chromatin alterations, we evaluated the relationship between these chromatin alterations and the drastic decrease or loss of sperm fertilizing ability after freeze-thawing. Using acridine orange staining and Feulgen-DNA quantitative microspectrophotometry, the nuclear variables of sperm were compared using samples which had either recovered, or lost their fertilizing ability after freezing-thawing during artificial inseminations, despite good post-thaw motility. There was a clear decrease in Feulgen-DNA content and nuclear surface values in sperm which exhibited a loss of fertilizing ability. Thus freeze-thawing may alter the DNA/nuclear protein relationships and impair the fertilizing ability of human sperm.

In vitro capacitation of human spermatozoa is commonly evaluated by the progressive motility percent. However its effects on sperm chromatin have hardly been studied. Our aim was to determine the extent to which in vitro capacitation with two treatments (B2 or human follicular fluid) alters the chromatin of human spermatozoa, by using two analytical methods, acridine orange staining and Feulgen-DNA cytophotometric measures. Ejaculates were obtained from 23 men participating in our in vitro fertilization program, and several measurements were made on the same ejaculate for each subject. No alteration was observed for the percent of native DNA after capacitation in B2, but spermatozoa incubation during the same time in human follicular fluid was followed by a significant decrease of the percent of native DNA (P less than 0.01). Feulgen-DNA content significantly increased after capacitation in either B2 or follicular fluid (P less than 0.05, P less than 0.001 respectively), and so did sperm nuclear surface area (P less than 0.001). In this study we observed a negative correlation between Feulgen-DNA content and fertilization rate (P less than 0.02). Moreover, the greater effects on Feulgen-DNA content were observed in men with abnormal sperm, whose spontaneous percent of native DNA was lower (P less than 0.05) and Feulgen-DNA content higher (P less than 0.05) than in men with normal sperm. These results indicate that capacitation in B2 as well as in human follicular fluid may alter the chromatin stability of human spermatozoa. Such results suggest a partial decondensation state of human spermatozoa during in vitro capacitation. However, beyond some level of decondensation, the fertilizing ability could be altered.

A case report of grave's neuropathy. The symptoms appeared in a treated patient when there was a biologic hypothyroidy. A total remission was observed after corticotherapy. Nevertheless, a recurrence of disease happened for reduced doses, so an orbital radiotherapy was requisited. Student of differents treatments for dysthyroid neuropathy.

Unlabelled: This article describes the outcome of the pregnancies conceived after AID with frozen semen in the Tours CECOS Centre between January 1st 1988 and December 31st 1986. There were 487 (including multiple pregnancies). These have been compared with a control group and with the national study conducted by INSERM in 1981. The study of the literature allows comparisons. The rate of pregnancies not resulting in a living child is not significantly different from the control series (abortions 11.4%, ectopic pregnancies 1% and in utero fetal deaths 0.4%). Pregnancies that continued: the rate of multiple pregnancies was higher (2.7%). There were more cases of bleeding (6.8% compared with 2.4%), more hypertensions (13.3% compared with 11.6%) and the same rate of premature labour (9.4%) as in the control group. The Caesarean section rate (22% as against 6%) was higher. The rate of forcepts delivery was lower (9.2% as against 12.8%). Concerning the children we found more premature babies (15.8% versus 5.6%) but no difference in the sex ratio, their height-weight as against their term, their Apgar score, morbidity or number or type of malformations (3.4%)., In Conclusion: the quality of the children is about the same with the proviso that the group is too small to conclude about the incidence of chromosomal abnormalities.

Functional ultrastructural changes in human sperm heads were evaluated following semen dilution in cryoprotective medium (with and without seminal plasma) as well as freezing/thawing. Plasma membranes were as much altered by dilution as by freezing/thawing when compared with fresh samples. The most striking effect on acrosome noted during freezing/thawing was a dramatic decline in percentage of intact spermatozoa. Acrosomal changes seemed to be less important in the fractions without seminal plasma. These deleterious effects were very demonstrable using transmission electron microscopy (TEM), rather than conventional staining. TEM is useful in obtaining more detailed information on membrane and acrosome integrity until a specific procedure to evaluate functional/physical integrity can be found. Cryopreservation in the absence of seminal plasma can be used for intrauterine insemination in AIH, AID programs with frozen semen.

Digital arterial circulation before and after administration of a vasodilator orally was explored by mercury gauge pulsed plethysmography and photoplethysmography in 12 patients with primary Raynaud's phenomenon and results compared with those of 10 healthy volunteers. The amplification factor F, ratio of amplitude in reactive hyperemia over amplitude at rest was determined in the index before and after 8 mg daily of dihydroergokryptine over 4 weeks. Before treatment, for each of the two plethysmographic technics, a significant increase in mean factor F values was noted in the patients with Raynaud's phenomenon when compared with healthy controls. This is due to a decline in digital arterial flow at rest. Using the mercury gauge plethysmograph, a significant reduction in factor F was observed after vasodilator treatment corresponding to an increase in digital flow at rest without increase in flow during hyperemia. Using photoplethysmography, no significant variation in factor F was noted after treatment. Mercury gauge plethysmography, which measures global digital blood flow appears to be a more sensitive method than photoplethysmography, which measures dermal and hypodermal blood flow, for follow-up of effects of vasodilator treatment on Raynaud's phenomenon.

Cryopreservation and freezing-thawing effects on the fertilizing ability of human spermatozoa commonly are evaluated by post-thaw motility. Various studies have depicted the ultrastructural changes caused by freezing-thawing, yet the chromatin alterations have been studied very limitedly. Our aim was to determine the extent to which freezing-thawing alters the chromatin of human spermatozoa, using two analytical methods: acridine orange staining and Feulgen-DNA cytophotometric studies. Both methods revealed a dramatic effect of freezing-thawing on sperm chromatin: the native DNA content decreased as did the Feulgen-DNA content, and sperm surface area was reduced. These results indicate an effect on DNA, diminished accessibility for Feulgen, and a decrease in nuclear surface area and prompt us to hypothesize a relationship between an "overcondensation" state for sperm chromatin after freezing-thawing and a lower conception rate for human semen after cryostorage.

Graves' disease was diagnosed in a 35-year old woman with a history of myxoedema in childhood. Clinical data, ultrasonography and radioisotope scanning showed that the disease had developed in an ectopic subhyoid thyroid gland. Although numerous thyroid diseases associated with an ectopic thyroid have been described, Graves' disease does not seem to be frequent since only one case has been published so far.

The observation of one patient with insulin resistance (114 unités of soluble insulin in 3 injections per day + 54 units of medium acting insulin at 8 pm) associated with a polycystic ovarian disease prompted us to discuss the relationship between hyperandrogenism, acanthosis nigricans and endogenous hyperinsulinism. An in vitro study of the insulin binding to patient's erythrocytes allowed to diagnose the rare Type A insulin-resistance syndrome proposed by Kahn.