Showing papers in "Human Reproduction in 2000"

Analysis of DNA fragmentation, plasma membrane translocation of phosphatidylserine and oxidative stress in human spermatozoa

Abstract: UNLABELLED The objectives of this cross-sectional observational study were: (i) to detect DNA damage and plasma membrane translocation of phosphatidylserine in purified sperm populations of high and low motility, and (ii) to analyse their relationship with the endogenous generation of reactive oxygen species. Ejaculates from infertile men were examined following gradient centrifugation. The main outcome measures were: sperm motion parameters (assessed with a computer analyser), generation of reactive oxygen species (measured by chemiluminescence), DNA damage (detected by terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling and monoclonal antibody labelling of single-stranded DNA) and translocation of membrane phosphatidylserine (examined with annexin V staining). DNA fragmentation and membrane translocation of phosphatidyl-serine were observed in the fractions with low and high sperm motility in all patients. The fractions with low sperm motility had significantly higher proportion of cells with DNA damage and production of reactive oxygen species than the fractions with high sperm motility (P < 0.005). DNA fragmentation was significantly and positively correlated with the generation of reactive oxygen species (r = 0.42; P = 0.02). IN CONCLUSION (i) spermatozoa from infertile men display translocation of membrane phosphatidylserine as diagnosed by annexin V positive staining; (ii) DNA damage (fragmentation and presence of single-stranded DNA) can be detected in ejaculated spermatozoa from infertile men in fractions with low and high sperm motility, and (iii) there is a relationship between DNA damage and oxidative stress.

The risks associated with pregnancy in women aged 35 years or older

Abstract: The obstetric risks of adverse outcome during pregnancy in women aged > or =35 years were quantified using a retrospective analysis of data from 385 120 singleton pregnancies in the North West Thames Region, UK, between 1988 and 1997. A comparison of pregnancy outcome was made on the basis of maternal age at delivery: 18-34 years (n = 336 462), 35-40 years (n = 41 327) and women aged > 40 years (n = 7331). Women aged 40 year old women, with adjusted odds ratios (OR) according to age group. Pregnant women aged 35-40 years were at increased risk of: gestational diabetes, OR = 2.63 [99% confidence interval (CI) 2.40-2.89]; placenta praevia = 1.93 (1.58-2.35); breech presentation = 1.37 (1.28-1.47); operative vaginal delivery = 1.5 (1.43-1.57); elective Caesarean section = 1.77 (1.68-1.87); emergency Caesarean section = 1.59 (1.52-1.67); postpartum haemorrhage = 1.14 (1.09-1.19); delivery before 32 weeks gestation = 1.41 (1.24-1.61); birthweight below the 5th centile = 1.28 (1.20-1. 36); and stillbirth = 1.41 (1.17-1.70). Women aged >40 years had higher OR for the same risks. Pregnant women aged >/=35 years are at increased risk of complications in pregnancy compared with younger women.

Sperm chromatin structure assay parameters as predictors of failed pregnancy following assisted reproductive techniques

Abstract: The predictive value of sperm chromatin integrity for pregnancy outcome following in-vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) was studied in 24 men attending a university-based assisted reproductive techniques laboratory using the flow cytometric sperm chromatin structure assay (SCSA). The SCSA is a measure of the susceptibility of sperm DNA to low pH-induced denaturation in situ. The mean percentage of spermatozoa in the neat sample demonstrating DNA denaturation was significantly lower in the seven men that initiated a pregnancy (15.4 +/- 4.6, P = 0.01) than in the 14 men who did not initiate a pregnancy (31.1 +/- 3.2). No pregnancies resulted if > or =27% of the spermatozoa in the neat semen sample showed DNA denaturation. These data demonstrate that SCSA parameters are independent of conventional semen parameters. Furthermore, the SCSA may allow physicians to identify male patients for whom IVF and ICSI will be unlikely to result in pregnancy initiation.

The morphology of human pronuclear embryos is positively related to blastocyst development and implantation

Abstract: Human embryos are selected for transfer using morphology at the cleaving and blastocyst stages. Zygote morphology has been related to implantation and pregnancy. The aim of this study was to relate pronuclear morphology to blastocyst development. Zygotes were scored according to distribution and size of nucleoli within each nucleus. Zygotes displaying equality between the nuclei had 49.5% blastocyst formation and those with unequal sizes, numbers or distribution of nucleoli had 28% blastocyst formation. Cleaving embryos that were selected initially by zygote morphology and secondarily by morphology on day 3 had increased implantation (IR) and pregnancy rates (PR; 31 and 57%), compared with those selected by morphology alone (19 and 33% respectively; P: < 0.01). There was a significant difference between zygote-scored and non-scored cycles on day 3 (PR: 57 versus 33%; IR: 31 versus 19%) and on day 5 (PR: 73 versus 58%; IR; 52 versus 39%). Zygote scoring can maintain pregnancy rates for both day 3 and day 5 transfers, increase implantation rates and reduce the numbers of embryos required to achieve a pregnancy.

Cytokine production by maternal lymphocytes during normal human pregnancy and in unexplained recurrent spontaneous abortion

Abstract: It has been proposed that successful pregnancy is a T helper 2-type phenomenon, and that T helper (Th)1-type reactivity is deleterious to pregnancy. The objective of this study was to compare the concentrations of Th1 and Th2 cytokines produced by peripheral blood mononuclear cells from women undergoing unexplained recurrent spontaneous abortion (RSA) with those produced during normal pregnancy at a similar gestational stage. The control group consisted of 24 women with a history of successful pregnancies and the abortion group comprised of 23 women with a history of unexplained RSA. Blood from the control group was obtained at the end of the first trimester as gestational age controls for the abortion group from whom blood was collected at the time of abortion. Phytohaemagglutinin-stimulated peripheral blood cell culture supernatants were analysed for concentrations of cytokines. Significantly higher concentrations of Th2 cytokines were produced by the first trimester normal group than by the RSA group, while significantly higher concentrations of Th1 cytokines were produced by the abortion group as compared to first trimester normal pregnancy, indicating a distinct Th2-bias in normal pregnancy and a Th1-bias in unexplained RSA.

Fertility and obstetric outcome after laparoscopic myomectomy of large myomata: a randomized comparison with abdominal myomectomy

Abstract: The purpose of this study was to compare, in infertile patients, the efficacy of laparoscopic myomectomy versus abdominal myomectomy, in restoring fertility and to evaluate the obstetric outcomes. Between January 1993 and January 1998, 131 patients of reproductive age, with anamnesis of infertility, underwent myomectomy because of the presence of at least one large myoma (diameter greater than or = 5 cm). Patients were randomly selected for treatment by laparotomy (n = 65) or laparoscopy (n = 66). The two groups were homogeneous for number, size and position of large myomata. Significant differences were found in the post-operative outcome: febrile morbidity (> 38 degrees C) was more frequent in the abdominal than in the laparoscopic group (26.2 versus 12.1%; P < 0.05). Laparotomy caused a more pronounced haemoglobin drop (2.17 +/- 1.57 versus 1.33 +/- 1.23; P < 0.001); three patients received a blood transfusion after laparotomy and none after laparoscopy. The post-operative hospital stay was shorter in the laparoscopic group (142.80 +/- 34.60 versus 75.61 +/- 37.09 h; P < 0.001). No significant differences were found between the two groups as concerns pregnancy rate (55.9% after laparotomy, 53.6% after laparoscopy), abortion rate (12.1 versus 20%), preterm delivery (7.4 versus 5%) and the use of Caesarean section (77.8 versus 65%). No case of uterine rupture during pregnancy or labour was observed.

Multiple gestation pregnancy

Abstract: The ESHRE Capri Workshop Group* rates in most circumstances. Embryo reduction involves extremely difficult decisions for infertile couples and should Correspondence should be addressed to Professor P.G. Crosignani, be used only as a last resort. Assisted reproductive treatClinica Ostetrica e Ginecologica I, Facolta de Medicina e Chirurgia, Universita Degli Studi di Milano, Via Commenda, ment centres and registries should express cycle results as 12-20122 Milano, Italy. E-mail: piergiorgio.crosignani@unimi.it the proportion of singleton live births; twin and triplet rates should be reported separately as complications of the Multiple gestation pregnancy rates are high in assisted procedures. Reducing the multiple gestation pregnancy reproductive treatment cycles because of the perceived need rate should be a high priority for assisted reproductive to stimulate excess follicles and transfer excess embryos in treatment programmes, despite the pressure from some order to achieve reasonable pregnancy rates. Perinatal patients to transfer more embryos in order to improve mortality rates are, however, 4-fold higher for twins and success. If nothing is done, public concern may lead 6-fold higher for triplets than for singletons. Since the goal to legislation in many countries, a step that would be of infertility therapy is a healthy child, and multiple unnecessary if assisted reproductive treatment programmes gestation puts that goal at risk, multiple pregnancy must be and registries took suitable steps to reduce multiple pregregarded as a serious complication of assisted reproductive nancy rates. treatment cycles. The 1999 ESHRE Capri Workshop

Treatment with the gonadotrophin-releasing hormone antagonist ganirelix in women undergoing ovarian stimulation with recombinant follicle stimulating hormone is effective, safe and convenient: results of a controlled, randomized, multicentre trial

Abstract: A multicentre, open-label, randomized study of the gonadotrophin-releasing hormone (GnRH) antagonist ganirelix (Orgalutran((R))/Antagon((TM))) was performed in women undergoing ovarian stimulation with recombinant FSH (rFSH: Puregon((R))). The study was designed as a non-inferiority study using a long protocol of buserelin (intranasal) and rFSH as a reference treatment. A total of 730 subjects was randomized in a treatment ratio of 2:1 (ganirelix:buserelin) using an interactive voice response system which stratified for age, type of infertility and planned fertilization procedure [IVF or intracytoplasmic sperm injection (ICSI)]. The median duration of GnRH analogue treatment was 5 days in the ganirelix group and 26 days in the buserelin group, whereas the median total rFSH dose was 1500 IU and 1800 IU respectively. In addition, in the ganirelix group the mean duration of stimulation was 1 day shorter. During ganirelix treatment the incidence of LH rises (LH >/=10 IU/l) was 2.8% versus 1.3% during rFSH stimulation in the buserelin group. On the day of triggering ovulation by human chorionic gonadotrophin (HCG), the mean number of follicles >/=11 mm diameter was 10.7 and 11.8, and the median serum oestradiol concentrations were 1190 pg/ml and 1700 pg/ml in the ganirelix and buserelin groups respectively. The mean number of oocytes per retrieval was 9.1 and 10.4 respectively, whereas the mean number of good quality embryos was 3.3 and 3.5 respectively. The fertilization rate was equal in both groups (62.1%), and the same mean number of embryos (2.2) was replaced. The mean implantation rates were 15.7% and 21.8%, and the ongoing pregnancy rates per attempt were 20.3% and 25.7% in the ganirelix and buserelin groups respectively. Evaluation of all safety data indicated that the ganirelix regimen was safe and well tolerated. The overall incidence of ovarian hyperstimulation syndrome was 2.4% in the ganirelix group and 5.9% in the reference group. The results of this study support a safe, short and convenient treatment regimen of ganirelix, resulting in a good clinical outcome for patients undergoing ovarian stimulation for IVF or ICSI.

Incidence of congenital malformations in children born after ICSI

Abstract: The aim of this study was to determine the incidence of congenital malformations in a complete cohort of children born after intracytoplasmic sperm injection (ICSI). The medical records were retrieved for 1139 infants, 736 singletons, 200 sets of twins and one set of triplets. The total number of infants with an identified anomaly was 87 (7.6%), 40 of which were minor. The incidence of malformations in children born after ICSI was also compared with all births in Sweden using data from the Swedish Medical Birth Registry and the Registry of Congenital Malformations. For ICSI children, the odds ratio (OR) for having any major or minor malformation was 1.75 [95% confidence interval (CI) 1.19-2.58] after stratification for delivery hospital, year of birth and maternal age. If stratification for singletons/twins was also done, the OR was reduced to 1.19 (95% CI 0.79-1.81). The increased rate of congenital malformations is thus mainly a result of a high rate of multiple births. The only specific malformation which was found to occur in excess in children born after ICSI was hypospadias (relative risk 3.0, exact 95% CI 1. 09-6.50) which may be related to paternal subfertility.

High frequency of sub-optimal semen quality in an unselected population of young men

Abstract: Male reproductive function seems to have deteriorated considerably during the past 4-5 decades. However, studies of the reproductive function in unselected populations have not previously been reported. As the large majority of young men in Denmark are subjected to a compulsory medical examination for military service, this provided a unique opportunity to study the reproductive function in an unbiased population. Altogether 891 young men delivered a blood sample in which reproductive hormones were measured. From 708 of these men data were also obtained on semen quality and testis size. The median sperm concentration was 41 x 10(6)/ml (mean 57.4 x 10(6)/ml). Men with ejaculation abstinence above 48 h had slightly higher sperm concentrations (median 45 x10(6)/ml, mean 63.2 x 10(6)/ml), but even in this subgroup, 21 and 43% respectively had sperm counts below 20 x 10(6)/ml and 40 x 10(6)/ml. Among men with no history of reproductive diseases and a period of abstinence above 48 h, as many as 18 and 40% respectively had concentrations below 20 and 40 x 10(6)/ml. Sperm counts were positively correlated with testis size, percentage normal spermatozoa and inhibin B, and negatively correlated with percentage immotile spermatozoa and follicle stimulating hormone. Possible causes for this high frequency of young men with suboptimal semen quality are obscure and need to be explored. Whether these findings apply for young male populations of comparable countries remains to be seen.

What does it mean to be a donor offspring? The identity experiences of adults conceived by donor insemination and the implications for counselling and therapy

Abstract: In the absence of research with adult donor offspring, this study begins to bridge that gap by asking individuals about their experiences as donor offspring and considering the implications for psychotherapeutic and counselling practice. Sixteen participants (13 male, three female, age range 26-55 years) recruited through donor insemination support networks in the UK, USA, Canada and Australia, were sent semi-structured questionnaires by E-mail and post. Using identity process theory as a framework for understanding participants' accounts, the data were qualitatively analysed using interpretative phenomenological analysis. Participants consistently reported mistrust within the family, negative distinctiveness, lack of genetic continuity, frustration in being thwarted in the search for their biological fathers and a need to talk to a significant other (i.e. someone who would understand). These experiences could be postulated as being indicative of a struggle to assimilate, accommodate and evaluate information about their new identities as donor offspring. Psychotherapists and counsellors need to be aware of these identity issues if they are to meet the needs of donor offspring within therapeutic practice.

Prospective randomized study of human chorionic gonadotrophin priming before immature oocyte retrieval from unstimulated women with polycystic ovarian syndrome

Abstract: The present study examined whether the rates of oocyte maturation, fertilization and development, as well as pregnancy rate could be improved by human chorionic gonadotrophin (HCG) priming 36 h before immature oocyte retrieval in patients with polycystic ovarian syndrome (PCOS). Immature oocyte retrieval was performed on day 10-14 of the cycles and patients were randomly allocated either to be primed with 10 000 IU of HCG before the retrieval, or not primed. Immature oocytes were cultured for 24-48 h in TC-199 medium with 20% (v/v) inactivated fetal bovine serum (FBS) supplemented with 75 mIU/ml follicle stimulating hormone (FSH) and luteinizing hormone (LH). Intracytoplasmic sperm injection (ICSI) was performed in all mature oocytes and the resulting embryos were transferred on day 2 or 3 after ICSI. A total of 17 patients underwent 24 completed treatment cycles. Thirteen cycles were primed with HCG and 11 other cycles were not primed. The mean number of oocytes retrieved was comparable in the two groups (7.8 +/- 3.9 versus 7.4 +/- 5.2). The percentage of oocytes achieving maturation at 48 h was significantly higher (P < 0.05) in the HCG-primed group (84.3%, 86/102) than in the non-HCG-primed group (69.1%, 56/81). Oocyte maturation was hastened in the HCG-primed group. Following 24 h of culture, 78.2 +/- 7.1% of oocytes were matured in the HCG-primed group compared with 4.9 +/- 2.5% of oocytes in the non-HCG-primed group (P < 0.001). There were no significant differences in the rates of oocyte fertilization and cleavage in these two groups. There were five clinical pregnancies (38.5%) in the HCG-primed group, and three pregnancies (27.3%) in the non-HCG-primed group.

Study of apoptotic DNA fragmentation in human spermatozoa

Abstract: The aim of our work was to define and better understand apoptosis in the spermatozoa of normal subjects, infertile patients and patients affected by specific tumoral diseases employing the method of the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling and confirming the results by electron microscopy. We studied 23 healthy, normozoospermic subjects (group A), 29 oligoasthenoteratozoospermic patients, affected by various andrological pathologies (group B), 28 patients with Hodgkin's disease (C1) and 30 patients with testicular cancer (C2). Our data demonstrate that the percentage of apoptosis in normozoospermic subjects (group A) is significantly lower than in all the other groups (B, C1, C2) (P < 0.001). This confirms that high DNA fragmentation is one of the characteristics of spermatogenetic failure. The induction of apoptosis, which can also be a basic response to neoplastic disease, can even act right up to the mature male gamete. Our results suggest that apoptosis could be the final result of various pathologies and of a deregulation of spermatogenesis control systems.

Differential mitochondrial distribution in human pronuclear embryos leads to disproportionate inheritance between blastomeres: relationship to microtubular organization, ATP content and competence

Abstract: It has been suggested that mitochondrial DNA defects that effect metabolic capacity may be a proximal cause of failures in oocyte maturation, fertilization, or early embryonic development. Here, the distribution of mitochondria was examined by scanning laser confocal microscopy in living human pronuclear oocytes and cleavage stage embryos, followed either by measurements of the net ATP content of individual blastomeres or anti-tubulin immunofluorescence to determine the relationship between mitochondrial distribution and microtubular organization. The results indicate that specific patterns of perinuclear mitochondrial aggregation and microtubular organization are related, and that asymmetrical mitochondrial distributions at the pronuclear stage can result in some proportion of blastomeres with reduced mitochondrial inheritance and diminished ATP generating capacity. While the inability to divide appears to be a development consequence for an affected blastomere, for the embryo, reduced competence may occur during cleavage if several blastomeres inherit a mitochondrial complement inadequate to support normal cellular functions. The findings provide a possible epigenetic explanation for the variable developmental ability expressed within cohorts of morphologically normal early cleavage stage human embryos obtained by in-vitro fertilization.

Ovarian stimulation with HMG: results of a prospective randomized phase III European study comparing the luteinizing hormone-releasing hormone (LHRH)-antagonist cetrorelix and the LHRH-agonist buserelin

Abstract: In this prospective and randomized study, 188 patients received the luteinizing hormone-releasing hormone (LHRH) antagonist cetrorelix, and 85 patients the LHRH agonist buserelin to prevent endogenous luteinizing hormone (LH) surges during ovarian stimulation in in-vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) cycles. Ultimately, 181 patients (96.3%) in the cetrorelix group, and 77 (90.6%) in the buserelin group, reached the day of the human chorionic gonadotrophin (HCG) injection. The mean number of human menopausal gonadotrophin (HMG) ampoules administered and the mean number of stimulation days with HMG were significantly less in the cetrorelix group than in the buserelin group (P < 0.01). A rise in LH and progesterone concentrations was observed in three of the 188 patients (1.6%) who received cetrorelix. On the day of the HCG administration, more follicles of a small diameter (11-14 mm) were observed in the buserelin group than in the cetrorelix group (P = 0. 02) and the mean serum oestradiol concentration was significantly higher in patients who received buserelin than in those who received cetrorelix (P < 0.01). Similar results were observed in fertilization, cleavage and pregnancy rates in the two groups. In conclusion, the use of the LHRH antagonists might be considered more advantageous because of the short-term application needed to inhibit gonadotrophin secretion, so allowing a reduction in the treatment time in a clinically significant manner.

Relationship between endometriotic foci and nerves in rectovaginal endometriotic nodules

Abstract: The histological relationships between fibrotic tissue, endometriotic foci and nerves in the rectovaginal septum endometriotic or adenomyotic nodule were studied. This is considered to be one of the most severe forms of deep endometriosis. Masson's trichrome staining for fibrosis detection and immunohistochemistry with the S100 monoclonal antibody for nerve detection were performed in 28 rectovaginal endometriotic nodules from patients presenting with severe dysmenorrhoea and deep dyspareunia (23 patients with no other endometriotic location or potential cause of pain at laparoscopy and ultrasonography; five patients with multiple pelvic endometriotic localizations and other potential causes of pain at laparoscopy). Patients were allocated to two groups on the basis of their preoperative pain scores for pelvic pain, dysmenorrhoea and deep dyspareunia (group 1, score >7; group 2, score < or =7). For each symptom, the mean number of nerves and endometriotic lesions per high-power field and the mean largest diameter of the lesions were not statistically different in groups 1 and 2. The mean percentages of nerves located within the fibrosis of the nodule and within endometriotic lesions were significantly higher in group 1 than in group 2. Among nerves located within endometriotic lesions, there was a significantly higher proportion showing intraneurial and perineurial invasion by endometriosis in group 1 than in group 2. In rectovaginal endometriotic nodules, there was a close histological relationship between nerves and endometriotic foci, and between nerves and the fibrotic component of the nodule. We postulate that such topographical relationships could at least partially explain the strong association between this lesion and pain.

Chromosome mosaicism in day 3 aneuploid embryos that develop to morphologically normal blastocysts in vitro

Abstract: In all, 143 human embryos obtained 3 days (day 3) after insemination or intracytoplasmic sperm injection (ICSI) were biopsied and a single nucleated cell removed for identification of aneuploidy by fluorescent in-situ hybridization (FISH) for chromosomes X, Y, 13, 16, 18 and 21. Fifty-one per cent of embryos were aneuploid and significantly more aneuploid embryos blocked in further development to morulae and blastocysts than euploid embryos (59 versus 34%; P < 0.001). Chromosomal analysis of the generated blastocysts revealed 40% were aneuploid (16 of 40 generated blastocysts). Re-examination of cells by FISH for the same chromosome probes of the inner cell mass (ICM) of expanded and hatching blastocysts derived from the aneuploid embryos revealed a high incidence of mosaicism of ICM cell lineages that were usually predictable from observations of day 3 single-cell biopsies. These data would not support the hypothesis of a preferential allocation of euploid cells to the ICM and aneuploid cells to the trophectoderm. A high concordance between day 3 aneuploidy diagnosis and ICM cell lineages was observed with trisomies (97%), and multiple complex chromosome numerical abnormalities (100%). A reduced concordance was observed with monosomies (65%) and haploidy (18%). Concomitantly, the proportion of ICM cell lineages was increased in blastocysts whose chromosomal condition was diagnosed as haploid (21%) or with complex numerical abnormalities (50%).

Transcription and replication of mitochondrial DNA

Abstract: The physical isolation of mammalian mitochondrial DNA (mtDNA) over 30 years ago marked the beginning of studies of its structure, replication and the expression of its genetic content. Such analyses have revealed a number of surprises: novel DNA structural features of the circular genome such as the displacement loop (D-loop); multiple sized and deleted forms of the circular genome; a minimal set of mitochondrially encoded rRNAs and tRNAs needed for translation; a bacteriophage-like, nuclear-encoded mitochondrial RNA polymerase for transcription; and a direct linkage between transcription and the commitment to replication of the leading mtDNA strand that centres on the nuclear encoded mitochondrial transcription factor A. One of the more recent revelations is the existence, near the D-loop, of an atypical, stable RNA-DNA hybrid (or R-loop) at the origin of mammalian leading-strand DNA replication, composed of the parent DNA strands and an RNA transcript. In mammalian mitochondrial systems, all of the proteins known to be involved in DNA replication are encoded in the nucleus. Thus alterations and deficiencies in mtDNA replication must arise from mutations in mtDNA regulatory sequences and nuclear gene defects. Further studies of the relationships between nuclear-encoded proteins and their mtDNA target sequences could result in strategies to manipulate genotypes within cellular mtDNA populations.

Cleavage anomalies in early human embryos and survival after prolonged culture in-vitro

Abstract: This study examines the relationship between common morphological anomalies of cleaving embryos and their ability to form apparently normal blastocysts in vitro. The impact of cleavage rate, fragmentation, and multinucleation on compaction, cavitation, along with inner cell mass and trophectoderm formation has been assessed. The study population consisted of 102 patients who elected or were selected to have a day 5 embryo transfer. Clinical pregnancy and implantation rates were 66.7 and 49% respectively. Slow and fast cleavage had a significant negative association with normal blastocyst formation. Only 13.8% (67/484) of embryos with 9 cells on day 3 formed blastocysts with apparently normal morphology, compared to 41.9% (252/602) with 7-9 cells on day 3 (P 15% fragmentation formed normal blastocysts at a significantly lower rate (46/279; 16.5%) than embryos with 0-15% fragmentation (311/935; 33.3%) (P < 0. 001). Furthermore, the pattern of fragmentation was associated with blastocyst formation. Type IV fragmentation led to a significant reduction in blastocyst formation (25/170 or 14.7%), compared to types I, II and III which performed much better (38.6, 32.9 and 32.4% respectively). Only 15.9% (22/138) of embryos with one or more multinucleate cells on day 2 and/or 3 formed normal blastocysts compared with 31.9% (335/1051) (P < 0.001) of those without multinucleation. Collectively, the data suggest that cleavage anomalies, some of which do not preclude development after short-term culture, may reduce the developmental competence of embryos after prolonged culture.

Prognostic value of Y deletion analysis: what is the clinical prognostic value of Y chromosome microdeletion analysis?

Abstract: In many centres, Y chromosome deletion analysis is still not performed routinely and if so, the results are used for genetic counselling but are not considered as having a useful prognostic value. The type of deletion (AZFa, b or c) has been proposed as a potential prognostic factor for sperm retrieval in men undergoing TESE. AZFc deletions and partial AZFb deletions are associated with sperm retrieval in approximately 50% of cases while in the case of a patient with complete AZFb deletion the probability of finding mature spermatozoa is virtually nil. Therefore the extent and position of a Y microdeletion is important (complete or partial). The prognostic value of Y chromosome deletion analysis in cases of oligozoospermia is important when one considers the progressive decrease of sperm number over time in men with AZFc deletions. Cryo-conservation of spermatozoa in these cases could avoid invasive techniques, such as TESE/ICSI, in the future. Male offspring that are conceived by ICSI or IVF techniques from father with oligozoospermia or azoospermia would also benefit from knowledge of their Y status, since the identification of the genetic defect will render future medical or surgical therapies unnecessary. Y microdeletion screening is therefore important, not only to define the aetiology of spermatogenic failure, but also because it gives precious information for a more appropriate clinical management of both the infertile male and his future male child.

Increased risk of early pregnancy loss by profound suppression of luteinizing hormone during ovarian stimulation in normogonadotrophic women undergoing assisted reproduction.

Abstract: The impact of suppressed concentrations of circulating luteinizing hormone (LH) during ovarian stimulation on the outcome of in-vitro fertilization or intracytoplasmic sperm injection treatment in 200 consecutive, normogonadotrophic women (couples) was analysed retrospectively. A standard stimulation protocol with mid-luteal gonadotrophin-releasing hormone (GnRH) agonist down-regulation and ovarian stimulation with recombinant follicle stimulating hormone (FSH) was used in all cases. Blood was sampled from each woman on stimulation days 1 and 8 for analysis of oestradiol and LH in serum. A threshold value of serum LH of 0.5 IU/l on stimulation day 8 (S8) was chosen to discriminate between women with low or 'normal' LH concentrations. Low concentrations of LH on S8 (<0.5 IU/l) were found in 49% (98/200) of the women. This group of women was comparable with the normal LH group with regard to pre-treatment clinical parameters, and to the parameters characterizing the stimulation protocol with the exception of serum oestradiol concentration, which on S8 was significantly lower than in the normal LH group (P < 0.001). The proportion of positive pregnancy tests was similar in the two groups (30% versus 34% per started cycle), but the final clinical treatment outcome was significantly different, with a five-fold higher risk of early pregnancy loss (45% versus 9%; P < 0.005) in the low LH group and consequently a significantly poorer chance of delivery than in the normal LH group. It is concluded that a substantial proportion of normogonadotrophic women treated with GnRH agonist down-regulation in combination with FSH, devoid of LH activity, experience LH suppression, which compromises the treatment outcome. Whether these women would benefit from supplementation with recombinant LH or human menopausal gonadotrophin during ovarian stimulation, remains to be proven in the future by prospective randomized trials.

Pregnancy outcome and deliveries following laparoscopic myomectomy

Abstract: Uterine rupture after myomectomy by laparotomy is not a common occurrence. Some case reports of uterine rupture after laparoscopic myomectomy (LM) raise the question of the quality of the uterine scar produced when this technique is performed. In order to assess the outcome of pregnancies and deliveries after LM and to assess the risk of uterine rupture, we performed an observational study. Questionnaires were mailed to all women who had had LM for at least one intramural or subserosal myoma of more than 20 mm diameter and who were aged <45 years. Ninety-eight patients became pregnant at least once after LM, giving a total of 145 pregnancies. Among the 100 patients who had delivery, there were three cases of spontaneous uterine rupture. Because only one of these uterine ruptures occurred on the LM scar, the risk of uterine rupture was 1.0% (95% CI 0.0-5. 5%). Seventy-two patients (72.0%) had trials of labour. Of these, 58 (80.6%) were delivered vaginally. There was no uterine rupture during the trials of labour. Spontaneous uterine rupture seems to be rare after LM. This risk should not deter the use of LM if needed. When performing LM, particular care must be given to the uterine closure.

Mitochondrial distribution and function in oocytes and early embryos

Abstract: Active mitochondria relocate during oocyte maturation or fertilization in several species. Detailed studies with hamster oocytes and early embryos reveal a pattern of active mitochondria migrating to surround the pronuclei to form a pattern that persists through the early cleavage stages. Although the functional significance of this relocation is unknown, it appears to be an important part of normal development in hamsters. Treatments that disrupt embryo development in vitro (such as the presence of inorganic phosphate or alteration of intracellular pH) also disrupt the normal pattern of mitochondrial distribution. Active mitochondria also reorganize during maturation in bovine oocytes and during fertilization in rhesus monkey oocytes. Examination of these changes in mitochondrial organization may provide insights into the regulation of normal embryo development and might serve as predictors of oocyte or embryo developmental competence.

Optimal use of infertility diagnostic tests and treatments. The ESHRE Capri Workshop Group.

Abstract: The general definition of infertility is a lesser capacity to conceive than the mean capacity of the general population and infertile couples can be characterized in two groups: those unable to conceive without therapy and those who are hypofertile, but conceive without therapy. The initial diagnostic tests for infertility should include a midluteal phase progesterone assay, a semen analysis and a test for tubal patency such as a hysterosalpingogram. Measuring progesterone is the best test for confirming ovulation. To predict ovulation, evaluating the luteinizing hormone (LH) surge is the best single assay while measurement of LH plus preovulatory oestrogen is the best prediction. Today primary investigation of the morphology of the uterus and tubes should be by hysterosalpingography. However, ultrasound, particularly with simple contrast media, is likely to gain in importance. Laparoscopy should be reserved as a further diagnostic procedure or in combination with endoscopic surgery. There are situations in which semen analysis is of utmost importance and of absolute predictive value, namely, in cases of azoospermia. In general semen analysis remains a substantial part of the fertility workup, but any consideration of its predictive value has to be cautious. Performing genetic tests before, during and after assisted reproductive techniques (ART) is an intrinsic part of good clinical practice. These tests allow one to reach a correct diagnosis, to give adequate genetic counselling to the couple and their families in cases such as (i) women with Turner syndrome; (ii) men with 47, XXY; (iii) men or women with structural chromosomal aberration; (iv) men with Yq11 deletion or (v) men with congenital bilateral absence of vas deferens. Patients should, of course, be made aware of the occurrence of de-novo mutations taking place in the testis and in the embryo. Treatment of some causes of infertility are of proven value. For example induction of ovulation. Others are more controversial. Among the many empirical treatments suggested for the treatment of the various form of subfertility, surgical treatment of varicocele in the male, treatment of pelvic endometriosis in the female and the efficacy of the ART strategies offered to the subfertile couple are considered. Many varicocele studies are of poor quality. A few are good, but small in size. They do not show an improvement in pregnancy rates. Therefore, at the moment, there is insufficient scientific evidence for recommending routinely surgical treatment in subfertile and/or oligozoospermic men with a varicocele. Randomized, double-blind controlled trials demonstrated the modest efficacy of endometriosis ablation in increasing the pregnancy rate in infertile women while drugs suppressing ovulation are of no benefit to infertile women with endometriosis. Although the largest body of evidence available suggests that IVF success declines in repeated ART cycles, an accurate estimate of the true success rate in the 'nth' cycle of IVF treatment is not possible. Similarly little is still known of the reasons for the overall low continuation rates with IVF treatment.

Computer-assisted semen analysis parameters as predictors for fertility of men from the general population

Abstract: The predictive value of sperm motility parameters obtained by computer-assisted semen analysis (CASA) was evaluated for the fertility of men from general population. In a prospective study with couples stopping use of contraception in order to try to conceive, CASA was performed on semen samples from 358 men. A recently developed CASA system, Copenhagen Rigshospitalet Image house sperm Motility Analysis System (CRISMAS) was used for assessment of motility parameters. This system has an editing function which allows correction of tracks made by the computer. Probably due to this function, the concentration assessment made by CRISMAS was very close to that made by the technician (median difference <5%) in all concentration ranges. Correlation between CASA parameters and fertility of normal couples (measured as probability of achieving pregnancy) was examined by the Cox regression model. In univariate models ln(sperm concentration) [beta = 0.331, risk ratio (RR) = 1.392, P = 0.0001], ln(total sperm count) (beta = 0.252, RR = 1.286, P = 0.0007) and percentage motile spermatozoa (beta = 0.014, RR = 1.014, P = 0.0004) were most significant predictors for fertility. In a multivariate analysis ln(sperm concentration) (beta = 0.268, RR = 1.307, P = 0.0016) and percentage motile spermatozoa (beta = 0.010, RR = 1.010, P = 0.011) but even more significantly the combined parameter, ln(concentration of motile spermatozoa) (beta = 0.329, RR = 1.389, P = 0.0001), were the only parameters of predictive value for fertility of men in the general population. In conclusion, these parameters obtained by CASA measurements can be used for prediction of fertility potential in normal men. This appears to be the first study showing the value of CASA in prediction of fertility in the general male population.

Increased risk of non-insulin dependent diabetes mellitus, arterial hypertension and coronary artery disease in perimenopausal women with a history of the polycystic ovary syndrome

Abstract: The aim of the study was to determine the prevalence of non-insulin dependent diabetes mellitus (NIDDM), arterial hypertension, coronary artery disease and the risk factors for these diseases in perimenopausal women with a history of polycystic ovary syndrome (PCOS) treatment A group of 28 women was selected from a large group of patients who had undergone wedge ovarian resection A total of 752 controls was selected by age (45-59 years) from a random female population sample There was no difference between the two groups in body mass index, waist circumference or waist-hip ratio Both groups were found to have identical family histories of NIDDM, hypertension, and coronary artery disease and identical smoking habits We did not find a difference between the mean concentrations of lipids and fasting glucose The two groups did not differ in the proportions of women with elevated lipid concentrations The prevalence of NIDDM and coronary artery disease was significantly higher in PCOS women In conclusion, women in the general population have the same level of risk factors at perimenopausal age as PCOS women Patients with markedly expressed clinical symptoms of PCOS made up a subgroup in the general population at high risk for developing NIDDM and coronary artery disease

Correlation between semen parameters and sperm aneuploidy rates investigated by fluorescence in-situ hybridization in infertile men.

Abstract: Spermatozoa from 32 infertile patients and 13 controls with normal semen parameters were analysed using dual and triple colour fluorescence in-situ hybridization (FISH) techniques, in order to investigate the rates of aneuploidy for chromosomes 13, 18, 21, X and Y. The patients were divided into three groups according to their karyotypes or the karyotypes of their offspring: 15 were infertile men with abnormal semen parameters and normal karyotypes (group 1), 13 were infertile men with abnormal karyotypes and normal or abnormal semen (group 2) and four were infertile men with abnormal semen and normal karyotypes but whose wives conceived a child (or a fetus) with a numerical chromosomal abnormality through an intracytoplasmic sperm injection cycle (group 3). Patients with abnormal semen parameters showed a significantly higher aneuploidy rate for the investigated chromosomes in their spermatozoa compared to controls (P < 0.005). Our data suggest the presence of a correlation between poor semen parameters and an increase in aneuploidy rate of chromosomes 13, 18, 21, X and Y in spermatozoa (r = -0.81071, P < 0.002); therefore the risk of a chromosomal aneuploidy in spermatozoa seems to be inversely correlated to sperm concentration and total progressive motility. Patients with abnormal karyotypes showed a higher incidence of diploidy and chromosomal aneuploidies compared to controls (P < 0.002). This strongly suggests the presence of an interchromosomal effect of the cytogenetic rearrangement. Men who fathered a child with an abnormal karyotype through intracytoplasmic sperm injection did not present a higher aneuploidy rate for the investigated chromosomes in spermatozoa compared to patients with infertility due to a similar male factor but showed higher incidence of chromosomal aneuploidy compared to normal controls.

Differences in nuclear DNA fragmentation and mitochondrial integrity of semen and prepared human spermatozoa

Abstract: Sperm DNA integrity is essential for accurate transmission of genetic material to offspring. Fragmentation of genomic DNA is an initial hallmark of apoptosis (programmed cell death). The aim of this study was to determine sperm nuclear DNA integrity and mitochondrial function, to quantify possible apoptosis and to investigate any relationship between these parameters. Semen samples (n = 25) were prepared by discontinuous Percoll density centrifugation (95.0:47.5). DNA integrity was determined using a modified alkaline single cell gel electrophoresis (Comet) assay. DNA fragmentation, possibly indicative of apoptosis, was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL). Mitochondrial transmembrane potential was determined using the mitochondrial probe 5,5',6,6'-tetrachloro-1,1', 3,3'-tetraethylbenzimidazolyl carbocyanine iodide (JC-1). The DNA integrity of prepared spermatozoa was significantly greater than that of semen (P < 0.005). Further, the percentage of spermatozoa with fragmented DNA and the degree of fragmentation within these cells in prepared spermatozoa is significantly less than in semen (P < 0.005). There is a significant correlation between DNA damage quantified using the Comet assay and DNA fragmentation determined using TUNEL (R = 0.562, P < 0.01). The percentage of spermatozoa with dysfunctional, possibly apoptotic, mitochondria was significantly lower in prepared spermatozoa than in neat semen samples (P < 0.001). There was a negative correlation between the percentage of spermatozoa with dysfunctional mitochondria and the percentage of progressively motile spermatozoa (R = -0.67, P < 0.01).

Mitochondrial morphology in human fetal and adult female germ cells.

Abstract: The aim of this study has been to observe, by electron microscopy, the morphological changes affecting mitochondria and associated organelles in the human female germ cell during oogenesis, maturation and fertilization. In the primordial germ cell (PGC), rounded mitochondria with a pale matrix and small vesicular cristae are disposed near the nucleus and significantly increase in number during PGC migration and settlement in the gonadal ridge, where they differentiate into oogonia. In these early stages of mammalian oogenesis, aggregates of mitochondria are typically clustered around or in close relationship with the nuage. In oocytes at early prophase stage, mitochondria proliferate while aligned along the outer surface of the nuclear membrane, contain a more dense matrix than before, and have lamellar cristae. Oocytes of primordial and primary follicles mostly contain round or irregular mitochondria whose matrix has become very light. These mitochondria show typical parallel, arched cristae, and are clustered near the nucleus with other organelles forming the Balbiani's vitelline body. When follicles grow, the mitochondria of the oocytes become even more numerous and are dispersed in the ooplasm. Both paranuclear accumulation and subsequent dispersion of mitochondria in the cytoplasm are likely to be regulated by microtubules. By ovulation, mitochondria are the most prominent organelles in the ooplasm. They form voluminous aggregates with smooth endoplasmic reticulum (SER) tubules and vesicles. These mitochondrial-SER aggregates (M-SER) and the mitochondrial-vesicle complexes (MV) could be involved in the production of a reservoir of substances or membranes anticipating subsequent fertilization and early embryogenesis. Just after fertilization, the mitochondria of the oocyte undergo a further substantial change in size, shape, and microtopography. In the pronuclear zygote, mitochondria concentrate around the pronuclei. During the first embryonic cleavage divisions, round or oval mitochondria with a dense matrix and few arched cristae are gradually replaced by elongated ones with a less dense matrix and numerous transverse cristae. A progressive reduction in size and number of M-SER aggregates and MV complexes also occurs. In summary, oocyte mitochondria show dynamic morphological changes as they increase in number and populate different cell domains within the oocyte. They form complex relationships with other cell organelles, according to the different energetic -metabolic needs of the cell during differentiation, maturation, and fertilization, and are ultimately inherited by the developing embryo, where they eventually assume a more typical somatic cell form.

The use of two density gradient centrifugation techniques and the swim-up method to separate spermatozoa with chromatin and nuclear DNA anomalies

Abstract: Human semen is heterogeneous in quality, not only between males but also within a single ejaculate. Differences in quality are evident, both when examining the classical parameters of sperm number, motility and morphology and in the integrity of the sperm nucleus. The aim of this study was to determine the efficiency of the PureSperm((R)), Percoll((R)) and swim-up preparation techniques to eliminate spermatozoa with nuclear anomalies. Semen samples were collected, washed and one part of the semen spread on a slide, the remainder was prepared using the swim-up, PureSperm((R)) or Percoll((R)) techniques. Spermatozoa from different fractions were fixed on slides and assessed. Sperm samples (n) from different men were stained using the chromomycin A(3) (CMA(3)) fluorochrome, which indirectly demonstrates a decreased presence of protamine (n = 31 for swim-up; n = 45 for PureSperm((R)); n = 39 for Percoll((R))). Spermatozoa prepared using PureSperm((R)) (n = 35) and Percoll((R)) (n = 37) were also examined for the presence of endogenous DNA nicks. Good quality spermatozoa should not possess DNA nicks and not stain (i.e. fluoresce) with CMA(3). When prepared using the swim-up technique the spermatozoa recovered showed no significant improvement with the CMA(3) staining. When spermatozoa were prepared using the PureSperm((R)) and Percoll((R)) techniques, a significant (P < 0.001) decrease in both CMA(3) positivity and DNA strand breakage was observed. These results indicate that both the PureSperm((R)) and Percoll((R)) techniques can enrich the sperm population by separating out those with nicked DNA and with poorly condensed chromatin.