Frozen-thawed embryo transfer: influence of clinical factors on implantation rate and risk of multiple conception.

BACKGROUND: Frozen embryo transfer is an important supplementary procedure in the treatment of infertility. While general information concerning the outcome of frozen embryo transfer has been documented, few studies have addressed the potential of embryo implantation in particular clinical situations. Importantly, the risk of multiple conception following frozen embryo transfer has been poorly documented compared with the information available for fresh embryo transfer. METHODS: This is a retrospective study analysing 3570 frozen embryo transfer cycles (1438 couples) with a view to increasing our understanding of the clinical circumstances that influence the potential for embryo implantation. RESULTS: The overall implantation rate was 9.1%. The characteristics associated with a more favourable implantation rate were the success of the previous fresh embryo transfer cycle, age < 40 years and non-tubal factor aetiology of infertility. Such women had an increased risk of multiple conception. CONCLUSION: Female age, the aetiology of infertility and the outcome of fresh embryo transfer are the most important factors influencing the implantation rate following frozen embryo transfer. A prognostic table has been constructed that may assist with the determination of the optimal number of embryos to be replaced in frozen embryo transfer to provide better individualized counselling and to secure an optimal chance of pregnancy while reducing the risk of multiple conception.

The coincidence of light and melatonin with a specific phase of the circadian pacemaker is important for the timing of seasonal breeding in the ewe.

The timing of reproductive activity in seasonal breeding sheep relies on daily photoperiodic signals being relayed to provide information on the time of year. Although light and melatonin are involved, the exact mechanism is not understood. In this experiment, three groups of 6 Romney Marsh ewes, a highly seasonal breed, were provided with 8 weeks of short nights (9.6-9.8 h, by artificially advancing dawn) around the winter solstice, near the end of their natural breeding season. One group of animals was infused to a physiological level with melatonin for 5 h during the afternoon prior to the onset of dark, while a second group was identically infused but for 5 h from the time of lights on. A third group received the short-night treatment only. Following the short-night treatment, all groups were exposed to long nights (> 14 h, by delaying dawn) until the summer solstice. Ovarian activity, assessed by progesterone monitoring twice weekly, showed that the noninfused and the morning-infused groups displayed renewed reproductive activity in response to the short-night/long-night treatment. There was no renewed ovarian activity in the afternoon-infused group, indicating that the time of day that melatonin is present, rather than the duration of melatonin exposure, is an important signal in the control of reproductive timing. Measurements of a marker of the endogenous circadian pacemaker, by melatonin measurements under acutely extended darkness, revealed that the short-night treatments phase advanced the onset of the pacemaker in all groups such that the afternoon phase of the pacemaker was coincident with light. The results provide strong support for the model that proposes that an afternoon-located sensitive phase of the pacemaker is responsible for the relay of photoperiodic signals in the timing control of seasonal breeding. The model proposes that the reproductive axis be primed during short nights when the sensitive phase is coincident with light in the afternoon so ovarian activity can be induced when the sensitive phase is located within the longer nights of autumn and coincident with endogenous melatonin.

FISH analysis of six chromosomes in unfertilized human oocytes after polar body removal.

PURPOSE: To develop an improved technique for estimating chromosomal abnormalities in human oocytes by fluorescence in situ hybridization (FISH) and to correlate the position of single chromatids with the chromosomal status of the oocytes. METHODS: Oocytes that were at metaphase II about 17-20 hr after insemination or intracytoplasmic sperm injection (ICSI) were treated with pronase to remove the zona pellucida and polar body (PB) and then spread on slides using HCl and Tween 20. Two rounds of FISH were performed using direct-labeled probes: chromosomes 1, 13, 21 (round 1); chromosomes X, 7, 18 (round 2). RESULTS: Of the 63 oocytes from 18 patients (mean age, 32 years), 48 (76%) had one DNA complement as expected, 9 (14%) had 2 DNA complements, 3 (5%) gave incomplete FISH signals, and 3 (5%) were not analyzable. Of the 48 oocytes with one set of DNA, 48% were haploid, 44% were aneuploid for one or more chromosomes, and 8% were polyploid. We also found an increased frequency of predivision of chromatid bivalents in aneuploid oocytes, especially for chromosome 21. CONCLUSIONS: This technique enables simultaneous assessment of six chromosomes in human oocytes, and therefore can be useful for accurately determining the incidence and causes of genetic imbalances in human oocytes and apparently low fertilization rates.

Congenital bilateral absence of the vas deferens, cystic fibrosis mutation analysis and intracytoplasmic sperm injection.

The aim of this study was to assess the outcome of intracytoplasmic sperm injection (ICSI) with fresh and frozen-thawed surgically retrieved spermatozoa from men diagnosed with congenital bilateral absence of the vas deferens (CBAVD). Twenty-seven azoospermic men with their partners were treated [25 with CBAVD and two with clinical cystic fibrosis (CF)]. CF gene mutation analysis and genetic counselling was provided. Spermatozoa were aspirated by microsurgical epididymal sperm aspiration (MESA), percutaneous epididymal sperm aspiration (PESA) or open testis biopsy. Of the men with CBAVD, 60% carried a single mutation, 20% were compound heterozygotes, and 20% had no CF mutation identified. Of the 28 sperm aspiration procedures, 86% had supplementary spermatozoa for cryopreservation with 83% of those samples assessed as satisfactory when thawed. Of 29 cycles with fresh spermatozoa a fertilization rate of 76% of oocytes injected and 17% embryo implantation rate occurred. Twenty-four cycles in which cryopreserved spermatozoa were used resulted in an oocyte fertilization rate of 69% and embryo implantation rate of 20%. Eighteen clinical pregnancies occurred with 14 live births without congenital anomaly. Two pregnancies were achieved following pre-implantation genetic diagnosis. It is concluded that the presence of CF mutations in the male partner does not compromise in-vitro fertilization treatment outcomes or the opportunity for healthy live births.

Analysis of five Duchenne muscular dystrophy exons and gender determination using conventional duplex polymerase chain reaction on single cells.

We have developed five conventional duplex polymerase chain reaction (PCR) protocols on single lymphocytes and blastomeres from embryos, in order to analyse five exons commonly deleted in deletion-type Duchenne muscular dystrophy (DMD). The five DMD gene exons (17, 19, 44, 45 and 48) can be analysed in separate duplex PCR reactions together with the sex-determining region Y (SRY) gene which enables simultaneous gender assignment. We present here PCR amplification results from single lymphocytes isolated from a normal male (220 cells), a normal female (24 cells) and a male DMD patient (40 cells) carrying a deletion of exons 46-49 within the DMD gene. The method failed to produce a PCR signal for the SRY gene in 8/220 normal male cells (3.6%) and for a DMD exon in 0-4.5% of normal male cells. One negative control out of 112 was positive. When this method was used to analyse two blastomeres from each of five embryos, concordant results were obtained for each pair of blastomeres. All embryos produced signals for the DMD exon tested with four of the embryos found to be male and one female. This method is therefore suitable for preimplantation genetic diagnosis and will allow the transfer of healthy embryos (both male and female) in families carrying DMD gene deletions involving at least one of the five exons 17, 19, 44, 45 and 48.

Fertilization failures and abnormal fertilization after intracytoplasmic sperm injection.

This study addresses the incidence of failed (0%) and suboptimal (<50%) fertilization after intracytoplasmic sperm injection (ICSI), variation in the ICSI fertilization rate for specific couples, and the causes of fertilization failure and abnormal fertilization after ICSI. Failed fertilization occurred in only 37 of 1343 cycles (3%). The risk of failure was highest (37%) when only one oocyte was injected, and was lowest (0.8%) when five or more oocytes were collected. The incidence of suboptimal fertilization and the variation in the fertilization rate were studied in 87 couples who each had three cycles of ICSI in which four oocytes were injected with ejaculated spermatozoa. Approximately 74% of these couples achieved >50% fertilization in every cycle. Only 26% of the couples had <50% fertilization in one or more cycles, and most of these (17%) had only a single cycle with suboptimal fertilization. Only four of the 87 couples (5%) had suboptimal fertilization in all three cycles. The difference between the maximum and minimum fertilization rate for a couple was used as an index of variation of the fertilization rate. It was found that 47 couples (54%) had 0-25% variation, 33 couples (38%) had 26-50% fertilization and only seven couples (8%) had >50% variation. The causes of failed and abnormal fertilization were studied in unfertilized and abnormally fertilized oocytes after staining with Hoechst 33342. In total, 1005 unfertilized oocytes were studied, of which 828 (82%) were still at metaphase II and 177 (18%) were activated. Most of the oocytes (83%) contained a spermatozoon and, in the majority of these oocytes, the sperm head was partially or completely decondensed. Hence, failure of oocyte activation was the principal cause of fertilization failure. A similar pattern was observed in activated, unfertilized oocytes, although there was a higher incidence of intact spermatozoa in these oocytes compared with metaphase II, unfertilized oocytes. Interestingly, 56% of the activated oocytes contained a decondensed sperm head which was not processed into a male pronucleus. A total of 169 abnormally fertilized oocytes was also studied. Two anomalies were found: digyny due to retention of the second polar body and its subsequent transformation into a third pronucleus, and abnormal pronuclear size and number.

Alterations of estrous activity in the ewe by circadian-based manipulation of the endogenous pacemaker.

The timing of reproductive activity in the seasonal breeding Romney Marsh ewe depends on the measurement of photoperiodic time. In this experiment, artificial light and dark signals are provided in a measured sequence at an inappropriate time of year to induce breeding out of phase with environmental photoperiod. The endogenous circadian responses and reproductive effects are documented. One group (Group A, control) of 6 Romney Marsh ewes was held in natural photoperiod throughout the experiment. For 8 weeks centered about the winter solstice (Stage 1), an additional 18 animals (Groups B, C, and D) were exposed to an artificial earlier dawn. Measurements of endogenous melatonin performed under acutely extended darkness confirmed a phase advance of the endogenous circadian pacemaker of the suprachiasmatic nucleus compared to control animals. In Stage 2, to the summer solstice (21 December), Group B animals were returned to natural photoperiod, Group C animals were subjected to an earlier artificial dusk, and Group D animals were subjected to an artificial delayed dawn. Melatonin measurements during Stage 2 confirmed that onset and offset times for Group C were earlier and that onset and offset times for Group D were delayed compared to corresponding times for Group B animals. Ovarian activity was monitored throughout. During Stage 2, Groups C and D commenced reproductive activity in mid-spring, and this continued until the experimental conditions changed. Groups A and B commenced reproductive activity at the normal timing in the subsequent autumn. Although not exclusive, these results are consistent with a coincidence model to explain the timing of seasonal breeding in this species with a dusk-located phase of the endogenous pacemaker sensitive to both light and melatonin. The temporal relationship between circadian alterations and the environmental photoperiod warrants further investigation as an explanation for seasonal breeding.

Analysis of unfertilized oocytes subjected to intracytoplasmic sperm injection using two rounds of fluorescence in-situ hybridization and probes to five chromosomes.

Chromosomal aberrations are the major cause of pre- and post-implantation embryo wastage and some studies suggest that half of all human conception have a chromosomal abnormality. Analysis of gametes provides information on the origin of these chromosomal aberrations. The purpose of this study was to develop a reliable multi-probe fluorescence in-situ hybridization (FISH) procedure that would enable us to investigate aneuploidy in unfertilized oocytes subjected to intracytoplasmic sperm injection (ICSI). Oocytes were spread with HCl and Tween 20 solution, and then two rounds of triple-probe FISH were performed on each oocyte using directly-labelled centromeric probes: chromosomes 1, 7, 15 (overnight hybridization); chromosomes 1, X, Y (2 h hybridization). After the first round, the slides were counterstained and evaluated, and the positions of FISH signals were recorded. For the second round, the counterstain was removed and the second probe cocktail was applied. The chromosome 1 probe was an internal control for the two hybridization procedures, while the Y chromosome probe was used to detect sperm DNA. To evaluate the method, a total of 79 oocytes from 27 patients were studied. Of these, 67 (84.8%) were successfully spread and 97% of these oocytes exhibited discernible FISH signals. Upon lysis, oocytes exhibited one or more DNA fragments (mean 1.9, range 1-3). Of the 65 analysable oocytes, 17 (26.2%) displayed a normal haploid chromosome constitution with paired spots for the two chromatids. A further 23 oocytes (35.4%) showed an ambiguous chromosome complement due to an abnormal number of DNA fragments which may have resulted from loss of DNA during spreading or to an abnormal oocyte, while 25 oocytes (38.4%) displayed aneuploidy for one or more of the chromosomes studied. In conclusion, this new approach is a quick and efficient method with which numerical chromosomal abnormalities in human oocytes can be studied; interpretation of the patterns of DNA fragments and FISH signals requires further clarification.

Estimation of aneuploidy for chromosomes 3, 7, 16, X and Y in spermatozoa from 10 normospermic men using fluorescence in-situ hybridization.

Fluorescence in-situ hybridization (FISH) is a fast and efficient method of estimating aneuploidy in human spermatozoa. In this study, we have estimated baseline disomy frequencies in spermatozoa from a group of 10 normospermic men, using stringent scoring criteria. A triple-probe FISH procedure was used for chromosomes 3, X and Y, while a double-probe FISH method was used for chromosomes 7 and 16. A total of 101273 spermatozoa were scored for chromosomes 3, X and Y, resulting in 97.83% haploidy (3X or 3Y), 0.39% disomy (33X, 33Y, 3XX, 3YY or 3XY) and 0.35% diploidy (33XX, 33YY or 33XY). A total of 100760 spermatozoa were scored for chromosomes 7 and 16, giving 98.9% haploidy (716), 0.11% disomy (7716 or 71616) and 0.27% diploidy (771616). Disomy frequencies for individual chromosomes differed (chromosome 3, 0.20%; chromosome 7, 0.05%, chromosome 16, 0.06%; X + Y, 0.19%). The frequency of disomy 3 was significantly higher than disomy 7 (P = 0.019) and disomy 16 (P = 0.022), while the frequency of sex chromosome disomy was significantly higher than disomy 7 (P = 0.0058) and disomy 16 (P = 0.0067), but not disomy 3 (P = 0.73). The disomy and diploidy (0.27-0.35%) estimates obtained for this normospermic population were generally low and were similar to other recent reports.

Detection of chromosomes and estimation of aneuploidy in human spermatozoa using fluorescence in-situ hybridization.

The development and application of fluorescence in-situ hybridization (FISH) has opened the way for comprehensive studies on numerical chromosome abnormalities in human spermatozoa. FISH can be rapidly applied to large numbers of spermatozoa and thus overcomes the major limitation of karyotyping spermatozoa after penetration of zona-free hamster oocytes. The simultaneous hybridization of two or more chromosome-specific probes to spermatozoa and subsequent detection of the bound probes using different fluorescent detection systems enables two or more chromosomes to be localized simultaneously in the same spermatozoon and provides a technique for undertaking reasonable estimates of aneuploidy. The most commonly used probes are those which bind to the centromeric region of specific chromosomes. Most studies to date have concentrated on estimating aneuploidy in spermatozoa from normospermic men, although reports are beginning to appear on aneuploidy in spermatozoa from subfertile and infertile men. Multi-probe FISH studies have generally reported disomy (hyperhaploidy) estimates of 0.05-0.2% per chromosome. There is preliminary evidence that some chromosomes such as X, Y and 21 are predisposed towards higher rates of non-disjunction during spermatogenesis. There are also suggestions of inter-donor variability in aneuploidy frequencies for specific chromosomes, although this requires confirmation in larger studies. While FISH is clearly a powerful technique that has many applications in reproductive medicine, it must also be realized that it does have limitations and the technology itself is still evolving and has yet to be fully validated on spermatozoa.

Failure of multitube sperm swim-up for sex preselection.

OBJECTIVE: To use double-label fluorescence in situ hybridization to evaluate a modified swim-up procedure that is purported to be effective for preconceptual sex selection. DESIGN: Controlled, blinded study. SETTING: University hospital laboratories. PATIENT(S): Donor males reporting for routine semen analysis. MAIN OUTCOME MEASURE(S): Percentages of X- and Y-bearing spermatozoa in neat semen and in two swim-up fractions, determined using double-label fluorescence in situ hybridization. RESULT(S): No clinically significant change from a 1:1 ratio was found in the distribution of X- or Y-bearing spermatozoa after double-label fluorescence in situ hybridization following a modified swim-up procedure and irrespective of the time (15, 30, 45, and 60 minutes) allowed for swim-up. CONCLUSION(S): Using fluorescence in situ hybridization, a modified swim-up procedure was evaluated for its purported ability to skew the relative percentages of X- and Y-bearing spermatozoa. No clinically significant change in the ratio of X- to Y-bearing spermatozoa was detected independent of time. Therefore, clinical application of this procedure should be strongly discouraged.

Albumin gradients do not enrich Y-bearing human spermatozoa.

The aim of this study was to evaluate objectively whether or not discontinuous albumin gradients enrich the proportion of Y-bearing human sperm. A blinded, collaborative trial design was employed whereby a licensed centre prepared the sperm fractions using licensed procedures, coded the sperm slides and then sent them to an independent laboratory for determination of the X:Y ratio in each sperm fraction using X and Y chromosome-specific probes and double label fluorescence in-situ hybridization (FISH). The identification codes and FISH results were collated by an independent third observer. Two albumin gradient methods which are currently used by licensed centres for male sex pre-selection, protocol 3 and modified protocol 3, were tested. Essentially the same results were obtained for the two methods. Highly motile sperm fractions were recovered from the albumin gradients, and the recoveries of motile spermatozoa (1.3-8.5%) were within the optimal range reported to produce maximal enrichment of Y-bearing spermatozoa. FISH analysis, however, revealed no enrichment for Y-bearing spermatozoa with either method, and the overall X:Y ratios were not significantly different from 1.0. Some samples showed marginal enrichment of Y-bearing spermaotozoa, whereas others showed marginal enrichment of X-bearing spermaotozoa. In conclusion, this collaborative study has demonstrated that the protocol 3 and modified protocol 3 albumin gradient procedures do not enrich Y-bearing spermatozoa. The clinical use of albumin gradients for male sex preselection should be reconsidered in the light of this and other evidence.

Preliminary observations on polar body extrusion and pronuclear formation in human oocytes using time-lapse video cinematography.

In this study, we have used time-lapse video cinematography to study fertilization in 50 human oocytes that had undergone intracytoplasmic sperm injection (ICSI). Time-lapse recording commenced shortly after ICSI and proceeded for 17-20 h. Oocytes were cultured in an environmental chamber which was maintained under standard culture conditions. Overall, 38 oocytes (76%) were fertilized normally, and the fertilization rate and embryo quality were not significantly different from 487 sibling oocytes cultured in a conventional incubator. Normal fertilization followed a defined course of events, although the timing of these events varied markedly between oocytes. In 35 of the 38 fertilized oocytes (92%), there were circular waves of granulation within the ooplasm which had a periodicity of 20-53 min. The sperm head decondensed during this granulation phase. The second polar body was then extruded, and this was followed by the central formation of the male pronucleus. The female pronucleus formed in the cytoplasm adjacent to the second polar body at the same time as, or slightly after, the male pronucleus, and was subsequently drawn towards the male pronucleus until the two abutted. Both pronuclei then increased in size, the nucleoli moved around within the pronuclei and some nucleoli coalesced. During pronuclear growth, the organelles contracted from the cortex towards the centre of the oocyte, leaving a clear cortical zone. The oocyte decreased in diameter from 112 to 106 microm (P < 0.0001) during the course of the observation period. The female pronucleus was significantly smaller in diameter than the male pronucleus (24.1 and 22.4 microm respectively, P = 0.008) and contained fewer nucleoli (4.2 and 7.0 respectively, P < 0.0001). After time-lapse recording, oocytes were cultured for 48 h prior to embryo transfer or cryopreservation. Embryo quality was related to fertilization events and periodicity of the cytoplasmic wave, and it was found that good quality embryos arose from oocytes that had more uniform timing from injection to pronuclear abuttal and tended to have a longer cytoplasmic wave. In conclusion, we have shown that time-lapse video cinematography is an excellent tool for studying fertilization and early embryo development, and have demonstrated that human fertilization comprises numerous complex dynamic events.

Application of modern molecular techniques to evaluate sperm sex selection methods.

The aim of this paper is to review modern approaches which have been used to evaluate sex pre-selection procedures. Two approaches can be used, polymerase chain reaction (PCR) and fluorescence in-situ hybridization (FISH). FISH is currently the method of choice for evaluating sex selection procedures because: (i) FISH accurately identifies the sex chromosome of individual spermatozoa using specific probes for the X and Y chromosomes and a two-colour detection system; and (ii) large numbers of spermatozoa can be screened in a short period of time. Of the published sex pre-selection methods tested using FISH, only flow cytometry has been shown to produce a clinically significant enrichment of X- and/or Y-bearing human spermatozoa. Studies have shown that 12-step Percoll gradients produce a slight but clinically insignificant enrichment of X-bearing spermatozoa, swim-up techniques do not appear to enrich either X- or Y-bearing spermatozoa, and discontinuous albumin gradients do not enrich Y-bearing spermatozoa. Despite this evidence, some of these methods continue to be used clinically, so it is vital that sex selection methods are properly evaluated using reliable methods such as double-label FISH before they are introduced for clinical use.

The influence of sperm morphology and the number of motile sperm inseminated on the outcome of intrauterine insemination combined with mild ovarian stimulation.

OBJECTIVE: To determine the influence of sperm morphology and the number of motile sperm inseminated on the outcome of IUI in hMG-stimulated cycles and to establish lower limits for these variables below which the expectation of pregnancy is limited. DESIGN: Retrospective study of data from 1990 to 1992. SETTING: Tertiary referral Reproductive Medicine Unit. PATIENTS: Couples with bilaterally patent fallopian tubes, and > or = 200,000 motile sperm recovered in a trial preparation before treatment. No other semen criteria were used to exclude couples. Women were stimulated with hMG irrespective of whether they were ovulatory or anovulatory. The study comprised 163 couples who underwent 330 cycles. MAIN OUTCOME MEASURES: Pregnancy rate (PR) per cycle was related to the percentage normal sperm morphology in the fresh semen sample and the number of motile sperm inseminated after sperm preparation by swim-up or Percoll gradients. RESULTS: The overall PR was 16.1% per cycle. The PR was highest in the first cycle of treatment (21.4%) and declined in the second and third cycles. The miscarriage rate was 10.4% and the incidence of multiple pregnancies was 13.9%. Two groups of patients were defined on the basis of sperm morphology: a "poor outcome" group ( < or = 10% normal) and a "good outcome" group ( > 10% normal). The PRs in these two groups were 4.3% and 18.2%, respectively, and the cumulative PRs after three cycles were 8.3% and 40.1%, respectively. The number of motile sperm inseminated did not significantly affect the PR. CONCLUSIONS: The degree of teratozoospermia affected the PR in hMG-stimulated IUI cycles and a normal morphology value of 10% in the fresh semen distinguished couples with good and poor outcomes. In contrast, the number of motile sperm inseminated did not significantly influence IUI outcome.

Nuclear structural conditions and PCR amplification in human preimplantation diagnosis.

An understanding of the relationship between nuclear morphology and DNA function is important in cytology and preimplantation diagnosis. In this study, direct polymerase chain reaction (PCR) amplification was used to diagnose the common delta F508 mutation of cystic fibrosis in 62 biopsied human embryo cells. The nuclei were photographed and classified into three categories depending on their microscopic appearance; these were further correlated with the results of PCR amplification. The normal nucleus group (42 embryo cells, with clear and regular nuclear membrane, transparent nucleoplasm and prominent nucleoli) showed 100% PCR amplification, with normal amplification results, i.e. bright DNA bands. These were considered to be the living cells. Only half of the cells (10 embryo cells) which contained abnormal nuclei (with abnormal nuclear membranes or nucleoplasm) showed PCR amplification, often with abnormal amplification results, i.e. weak DNA bands. These cells were considered to be either degenerate or to be undergoing degeneration. The anuclear cells (10 embryo cells) were composed of living (metaphase) and degenerated cells and showed about 30% PCR amplification. These results demonstrated that one of the important signs of early visible cell degeneration is the partial or total degeneration of the nucleus. Abnormal morphological changes of the nuclear membrane and nucleoplasm are usually accompanied with functional and structural DNA alteration. It is suggested that base degradation occurs earlier than the breakage of base-sugar bonds and phosphodlester bonds during the course of DNA degradation. The selection of optimal cells with a normal nucleus for single cell embryo biopsy is important for the precision and safety of preimplantation diagnosis.

Is a critical interval of the circadian pacemaker at dusk responsive to light and melatonin responsible for the timing of estrus in the Romney Marsh ewe?

Two experiments, using Romney Marsh ewes, tested for the existence and role of a critical interval of the circadian pacemaker located near dusk that may be integrally involved in the precise timing of the breeding season. Groups of Romney Marsh ewes (n = 6) were provided with exogenous melatonin by injection at dusk (Experiment 1) or by infusion at dawn or subjected to extended darkness at dawn (Experiment 2) from the winter to the summer solstice before being exposed to natural photoperiod at latitude 35 degrees S. Other than the experimental protocols, all animals were held in natural photoperiod. The onset of the breeding season (defined as cyclic ovarian activity as indicated by plasma progesterone monitoring) was normal in those animals treated with morning melatonin but was delayed in those animals treated with melatonin at dusk or extended darkness at dawn compared to controls in natural photoperiod (p < .01). Exogenous melatonin at dusk was associated with a phase advance of the onset of the circadian pacemaker (as measured by endogenous melatonin in acutely extended darkness); additional darkness at dawn was associated with a phase delay of both the onset and the offset of the circadian pacemaker. Exogenous morning melatonin did not change the phase of the circadian pacemaker relative to the controls. The results are consistent with an external coincidence model of seasonal breeding in which a critical interval of the circadian pacemaker requires exposure to light during spring/summer to time estrus correctly. The proposed critical interval appears to be located near dusk in this model and is phase locked to the circadian pacemaker. The effect of the exogenous melatonin on the timing of the breeding season is similar to darkness when administered at dusk but is not equivalent to darkness at dawn. The timing of anestrus was not affected by any of the experimental treatments and may reflect a common response to an environmental influence.

Optimal polymerase chain reaction amplification for preimplantation diagnosis in cystic fibrosis (delta F508)

OBJECTIVE: To evaluate direct polymerase chain reaction amplification of mutation on single embryo cells for the routine preimplantation diagnosis of cystic fibrosis. DESIGN: Direct polymerase chain reaction amplification of mutation was performed to identify the cystic fibrosis delta F508 mutation in human blood DNA, single lymphocytes, embryos, and embryo cells obtained by biopsy. Preimplantation diagnosis was performed for a couple who were heterozygous carriers of the delta F508 mutation. SETTING: Laboratory for preimplantation diagnosis in a reproductive medicine unit. MAIN OUTCOME MEASURE: Correct diagnosis of homozygous normal, heterozygous, and homozygous abnormal DNA of the cystic fibrosis delta F508 mutation. RESULTS: 45 blood samples (18 homozygous normal, 17 heterozygous, and 10 homozygous abnormal) and 204 single lymphocytes from known sources showed 100% amplification and were diagnosed correctly. 17 human embryos and 52 normal nucleated embryo cells obtained by single cell embryo biopsy also showed 100% amplification. After a miscarriage of the initial pregnancy (diagnosed at preimplantation to be homozygous normal) in the heterozygous carrier couple, fetal tissue was confirmed to be homozygous normal. CONCLUSION: Direct polymerase chain reaction amplification of mutation is a simple, fast, reliable test for the common cystic fibrosis mutation (delta F508) in blood DNA and single cells and should be applicable to routine programmes of general screening, maternal blood examination, and preimplantation diagnosis.