Gene expression analysis of a new source of human oocytes and embryos for research and human embryonic stem cell derivation.

OBJECTIVE: To create developmentally competent embryos from failed-to-fertilize oocytes for use in infertility research and human embryonic stem cell derivation. DESIGN: Attempts to recover developmental potential of failed-to-fertilize oocytes were made by using either parthenogenetic activation or reinsemination by intracytoplasmic sperm injection. Resulting embryos were cultured to various stages up to and including blastocyst, and single embryos exhibiting normal development were analyzed for gene expression by quantitatively profiling representative transcripts. SETTING: Hospital-based assisted reproductive technology laboratory and University academic laboratories. PATIENT(S): One hundred sixty-five couples undergoing assisted fertility treatment. INTERVENTION(S): Metaphase II stage oocytes were either parthenogenetically activated or reinseminated with donor sperm, then allowed to develop up to and including the blastocyst stage. MAIN OUTCOME MEASURE(S): Gene expression analysis was performed on oocytes and embryos by quantitative reverse transcriptase-polymerase chain reaction for markers of developmental competence. RESULT(S): Fertilization occurred in 65% of the activated or reinseminated oocytes, which resulted in a blastocyst formation rate of 8%. Evaluation of a number of developmentally important genes in those embryos exhibiting normal development revealed profile and levels of expression similar to control embryos. One blastocyst from an activated oocyte yielded a novel pluripotent stem cell line indistinguishable from those derived from embryos surplus to infertility treatment. CONCLUSION(S): Clinically unusable oocytes represent a valuable alternative source of normal human embryos for human infertility and stem cell research without conflicting with patient treatment.

Reducing the incidence of twins from IVF treatments: predictive modelling from a retrospective cohort.

BACKGROUND: IVF treatments carry a high risk of twin pregnancy which confers a higher risk to the mother and child than singletons. Increased use of elective single embryo transfer (eSET) can reduce this twin rate. We aimed to utilize a previously published data set and statistical model based on routinely collected clinical data to predict the outcomes of policies that increase the proportion of eSET. METHODS: The models allow simultaneous prediction of outcomes from double embryo transfer (DET) and SET. These models were used to predict outcomes for different scenarios using SET in both the initial (fresh) transfer and over a complete cycle (transfer of all embryos created, with cryopreservation). A total of 16 096 cycles (12 487 fresh and 3609 frozen) from 9040 couples treated between 2000 and 2005 were included in the final analyses. RESULTS: For any transfer, SET has about a one-third lower live birth rate relative to DET: this can be partially mitigated by appropriate patient and treatment cycle selection, with several realistic policies performing similarly. However, if we consider complete cycles with embryo cryopreservation, it is possible for repeat SET to produce more live births per egg retrieval than repeat DET. CONCLUSIONS: All patients receiving SET would have a higher chance of successful treatment in that cycle if they received DET. The selection of appropriate patients for SET can partially ameliorate the overall loss. For complete cycles, repeat SET could produce more live births per egg retrieval than repeat DET. All treatments involving SET will increase the number of treatments required to achieve a successful outcome and this extra treatment burden will be a significant barrier to the implementation of such treatments.

Clinically failed eggs as a source of normal human embryo stem cells.

The promise of human embryo stem cells (hESCs) for regenerative medicine is offset by the ethical and practical challenges involved in sourcing eggs and embryos for this objective. In this study we sought to isolate an hESC line from clinically failed eggs, the usage of which would not conflict with donor interests to conceive. A total of 8 blastocysts were allocated for hESC derivation from a pool of 579 eggs whose fertilization had been clinically assessed to have occurred abnormally (i.e., three pronuclei) or failed (i.e., no pronuclei) following in vitro insemination or intracytoplasmic sperm injection (ICSI). The latter were subjected to a recovery intervention consisting of either reinsemination by ICSI or parthenogenetic stimulation. One hESC line (RCM1) was obtained from a failed-to-fertilize inseminated egg recovered by parthenogenetic activation. Standard in vitro and in vivo characterization revealed this line to possess all of the properties attributed to a normal euploid hESC line. Whole-genome single-nucleotide polymorphism analysis further revealed that the line was biparental, indicating that sperm penetration had occurred, although parthenogenetic stimulation was required for activation. Our results demonstrate the viability of an alternative strategy to generate normal hESC lines from clinically failed eggs, thereby further minimizing the potential to conflict with donor reproductive interest to conceive.

Factors affecting successful outcome from ICSI in men following previous vasectomy.

There are conflicting reports as to whether the interval between vasectomy and surgical sperm retrieval (SSR) for intra-cytoplasmic sperm injection (ICSI) is related to clinical pregnancy (CPR), and live birth (LBR), rates. This study aimed to evaluate factors that may influence the outcome of ICSI in males with secondary azoospermia due to previous vasectomy. We analysed the medical records of 198 azoospermic males following vasectomy who underwent percutaneous epididymal sperm aspiration (PESA) and/or testicular sperm extraction (TeSE), between 1997 and 2005 by a single urologist, and whose sperm was subsequently frozen for use in an IVF treatment programme on their partner's behalf. Hundred and forty-four (73%) males had a positive PESA, and the remaining 54 (27%) had a positive TeSE. Forty-four percent of males with no clinical evidence of epididymal distension still had epididymal sperm retrieved successfully. Hundred and twenty-eight patients proceeded with ICSI, and a total of 237 cycles were performed. The CPR and LBR overall were 29 and 27%, respectively. Using logistic regression there was no association between time since vasectomy and CPR (P = 0.17) or LBR (P = 0.31). A history of an attempted reversal of vasectomy did not negatively affect retrieval rates or CPR and LBR. The success of SSR and the outcome of ICSI, using frozen sperm, are independent of male age and time since vasectomy. Epididymal sperm may be retrieved in over 40% of men in whom there is no clinical evidence of epididymal distension.

Sperm retrieval rates in subgroups of primary azoospermic males.

OBJECTIVES: Men presenting with primary infertility and azoospermia may be offered surgical sperm retrieval (SSR) as a prelude to intracytoplasmic sperm injection (ICSI). We evaluated sperm retrieval rates in subgroups of men with azoospermia, based on obstructive aetiology, testicular volume and FSH. METHODS: 106 patients with primary infertility underwent clinical evaluation and SSR with percuataneous epididymal aspiration (PESA) and/or testicular sperm extraction (TeSE) by a single urologist over a five year period. Ten percent of this group (11 patients) had a clear cause of obstruction, congenital absence of the vas deferens (CBAVD), labelled group A. Ninety percent (95 patients) had no definite cause of obstruction, labelled group B. RESULTS: All eleven patients in group A had adequate sperm retrieved, compared with 56% of 95 men in group B. Clinical pregnancy and live birth rates were 47% and 44% for group A respectively compared with 21% and 20% for group B. Twenty-one men had testes <4 cm and FSH>10; a significantly lower sperm retrieval rate was seen in this subgroup (29%) compared to men with normal testicular volume and FSH (77%), p=0.0001, which corresponded to a LBR of 28% and 14% respectively. CONCLUSIONS: In the absence of testicular histology prior to SSR clinical parameters can be used to aid in counselling. Azoospermic males with normal sized testes and normal FSH can expect acceptable numbers of sperm to be retrieved by SSR for ICSI. Less than one third of men with raised FSH and small testes will have successful SSR.

The optimal length of 'coasting protocol' in women at risk of ovarian hyperstimulation syndrome undergoing in vitro fertilization.

Ovarian hyperstimulation syndrome (OHSS) is a serious and potentially life-threatening complication following ovarian stimulation for in vitro fertilization (IVF). Coasting is the practice whereby the gonadotrophins are withheld and the administration of human chorionic gonadotrophin (hCG) is delayed until serum oestradiol (E2) has decreased to what is considered to be a safe level, to prevent the onset of OHSS. This study aimed to assess the length of coasting on the reproductive outcome in women at risk of developing OHSS. Coasting was undertaken when the serum E2 concentrations were > or = 17000 pmol/L but < 21000 pmol/L. Daily E2 measurements were performed and hCG was administered when hormone levels decreased to < 17000 pmol/L. Eighty-one women who had their stimulation cycles coasted were grouped according to the number of coasting days. Severe OHSS occurred in one case, which represented 1.2% of patients who underwent coasting because of an increased risk of developing the syndrome. No difference was found between cycles coasted for 1 - 3 days and cycles coasted for > or = 4 days in terms of oocyte maturity, fertilization and embryo cleavage rates. Women in whom coasting lasted for > or = 4 days had significantly fewer oocytes retrieved (P < 0.05) and decreased implantation rate (P < 0.05) compared to those coasted for 1 - 3 days. Pregnancy rate/embryo transfer and live birth rate did not differ between groups. In conclusion, coasting appears to decrease the risk of OHSS without compromising the IVF cycle pregnancy outcome. Prolonged coasting is, however, associated with reduced implantation rates, perhaps due to the deleterious effects on the endometrium rather than the oocytes.

Ovarian response to gonadotropins after laparoscopic salpingectomy or the division of fallopian tubes for hydrosalpinges.

OBJECTIVE: To compare the effect of prophylactic laparoscopic salpingectomy versus division of the fallopian tubes on ovarian response to gonadotropins in women undergoing IVF. DESIGN: Retrospective study. SETTING: National Health Service-based tertiary referral center for reproductive medicine. PATIENT(S): One hundred sixty-eight women with tubal factor infertility. Sixty-five women with hydrosalpinges had either salpingectomy (n = 40, group A) or proximal tubal division (n = 25, group B), while the remaining women with tubal disease but without hydrosalpinges acted as the control group (n = 103, group C). INTERVENTION(S): Prophylactic laparoscopic salpingectomy or proximal division of the fallopian tubes and ovarian stimulation with gonadotropins for IVF. MAIN OUTCOME MEASURE(S): Day 2 serum FSH levels before surgery and 3 months after surgery but before ovarian stimulation, ovarian response assessed as total dose of hMG administered, serum E2 concentrations on day 3 and day 8 of stimulation and on the day of hCG injection, number of follicles, and number of oocytes retrieved and fertilized. RESULT(S): In group A, baseline FSH levels were significantly raised after surgery compared with before surgery. Postsurgery FSH concentrations were significantly higher in group A compared with group B. The number of follicles (15-20 mm) was significantly lower in group A compared with group B and group C. The serum E2 levels on day 8 of stimulation were lower in group A compared with group B, and on the day of hCG injection it was significantly reduced in group A compared with groups B and C. The number of oocytes retrieved per cycle was significantly lower in group A compared with group B. There were no significant differences in pregnancy rates and miscarriage rates among the three groups. CONCLUSION(S): These findings suggest that prophylactic salpingectomy in women with hydrosalpinx may compromise ovarian response to stimulation without affecting pregnancy rates. A randomized control trial is recommended to determine the most appropriate laparoscopic procedure in the management of hydrosalpinx before IVF.

Cryopreserved-thawed embryo transfer in natural or down-regulated hormonally controlled cycles: a retrospective study.

OBJECTIVE: To assess the implantation, pregnancy, and live birth rates after the transfer of frozen-thawed embryos (FET) in a natural or hormonal control cycle. DESIGN: Retrospective study. SETTING: National Health Service tertiary referral center for reproductive medicine in Manchester, United Kingdom. PATIENT(S): Two comparable groups of women with regular menstrual cycles: Group A (n = 212) had FET in a natural cycle after spontaneous ovulation; group B (n = 205) had FET in a pituitary-desensitized hormonally controlled cycle. INTERVENTION(S): In group B, GnRH agonist was commenced in the midluteal phase of the previous cycle and discontinued 3 days before embryo transfer. Oral estradiol valerate and vaginal progesterone pessary were used to prepare the endometrium. Embryo transfer was carried out 3 days after detection of the endogenous LH surge in group A and on day 3 of progesterone administration in group B. MAIN OUTCOME MEASURE(S): Implantation, pregnancy, and live birth rates per cycle and per embryo transfer (ET). RESULT(S): In the 212 women who had natural-cycle FET, 172 ETs were performed and 247 embryos replaced. The implantation rate was 14.1% (35/247). Twenty clinical pregnancies (20/172, 11.6%) were achieved. In the 205 women who had down-regulated hormone replacement-cycle FET, 173 embryo transfers were performed and 243 embryos replaced. The implantation rate was 13.5% (33/243). Eighteen clinical pregnancies (18/173, 10.2%) were achieved. There were no significant differences between the two groups with regard to the implantation, clinical pregnancy, or live birth rates per cycle or per ET. CONCLUSION(S): These findings suggest that both FET protocols are equally effective in terms of implantation rate and pregnancy outcome in women with regular menstrual cycles.

Characterizing the endometrium in unexplained and tubal factor infertility: a multiparametric investigation.

OBJECTIVE: To characterize endometrial development in unexplained and tubal factor infertility. DESIGN: Prospective study of 20 women with unexplained infertility, 22 with tubal factor infertility, and 21 fertile controls in the midproliferative, periovulatory, and midluteal phases of the menstrual cycle. SETTING: Reproductive Medicine Department of St. Mary's Hospital, Manchester, United Kingdom. PATIENT(S): Women awaiting assisted conception. INVESTIGATION(S): Serum hormone assays, transvaginal ultrasound, Doppler, and midluteal endometrial biopsies. MAIN OUTCOME MEASURE(S): Serum levels of E2, P, and LH, endometrial ultrasound morphometry, uterine and subendometrial artery Doppler, and endometrial histology and biochemistry. RESULT(S): Women with unexplained infertility demonstrated significantly reduced uterine artery flow velocity in all phases, significantly elevated uterine and subendometrial artery impedance in the periovulatory and midluteal phases, and significantly reduced endometrial texture in the midproliferative phase. Women with tubal factor infertility demonstrated significantly reduced uterine artery flow velocity, without a concomitant increase in impedance, and significantly greater expression of endometrial glandular and luminal keratan sulphate. CONCLUSION(S): Unexplained infertility is associated with a profound impairment of endometrial perfusion that might be amenable to treatment by perfusion enhancers. Tubal factor infertility is associated with endometrial developmental defects that might be corrected by salpingectomy. Endometrial ultrasound and Doppler studies are likely to become a vital tool in the investigation of infertility.

Expression of 11 members of the BCL-2 family of apoptosis regulatory molecules during human preimplantation embryo development and fragmentation.

Apoptosis during preimplantation development has received much interest because of its potential role in eliminating defective cells. Although development in humans is characterised by a high degree of genetic abnormality, little is known of the regulation of apoptosis in embryos. By PolyA PCR we analysed expression of 11 BCL-2 genes in individual human embryos representative of normal development and in severely fragmented embryos. We demonstrate constitutive expression of BAX in virtually all embryos at all stages of development, and variable expression of BCL2, BCL-XL, BCL-W, MCL-1 BAK, BAD, BOKL, BID, BIK, and BCL-XS. The frequency of expression of pro- and anti-apoptotic BCL-2 members was similar throughout development, except at the two-cell stage where pro-apoptotic genes predominated. Protein expression was confirmed for BCL-2, MCL-1, BCL-X, BAX, BAD, and activated caspase 3. BCL-2 protein was associated with mitochondria but expressed inconsistently in the blastocyst inner cell mass. Consistent differences between morphologically intact and fragmented embryos included the expression of BAK in fragmented but not intact four-cell embryos. Our study addresses the importance of examining single human embryos representative of the viable population for a large number of genes, in order to establish meaningful expression profiles and provide information on overlapping function in a large gene family.

Ultrastructural preservation of ovarian cortical tissue cryopreserved in dimethylsulfoxide for subsequent transplantation into young female cancer patients.

Cytotoxic therapy in young women with cancer may cause loss of ovarian function, and ovarian cryopreservation has been proposed as a means of preserving fertility. The objective of this study is to assess ultrastructural preservation of follicles in frozen-thawed ovarian cortical strips, previously cryopreserved using dimethylsulfoxide and a standard slow-cool/rapid-thaw protocol. Ovarian cortical strips (patients, n=7) were thawed and fixed in glutaraldehyde for epoxy resin embedding electron microscopy according to conventional procedures. Oocytes were generally well preserved, on the basis of plasma-membrane integrity, uniformity of chromatin pattern, mitochondrial and rough endoplasmic reticulum (rER) cisternal integrity, and absence of cytoplasmic and intranuclear clear spaces. Frequently, rER cisternae were distended and intramitochondrial matrices lost. Granulosa cells showed somewhat variable preservation. Some were dark-staining; others exhibited washed-out cytoplasm containing damaged or artifactually expanded organelles. The consistently good preservation of oocytes and the good but more variable preservation of granulosa cells provides scientific validation of one component in the overall clinical procedure of attempting to reestablish fertility in young female patients after cytotoxic therapy for cancer.

Waiting for in vitro fertilization treatment: spontaneous and ART live births.

This study analysed the live birth rates in 760 couples referred in 1994 to St Mary's Hospital, Manchester, a non-fee-paying National Health Service (NHS) centre, who had waited for up to 4 years for IVF treatment. These live birth rates were compared with those of 199 couples referred at a similar time to Manchester Fertility Services, a fee-paying unit, where they received IVF treatment shortly after referral. The waiting time was advantageous in that 17.8% (135 of 760) of the couples referred to St Mary's Hospital conceived without IVF treatment, 60% within one year of referral. However, the waiting time was detrimental to women aged 30-34 in whom treatment was delayed by 3-4 years. Only 26.8% (204 of 760) of couples originally referred eventually received NHS-funded IVF treatment at St Mary's. A waiting time not exceeding 18 months would allow most spontaneous conceptions and reduce the adverse effect of prolonged waiting on the take-up rate for treatment and on the chance of success in the older women.

Amplification of representative cDNA pools from single human oocytes and pronucleate embryos.

In the human embryo, gene expression studies have been hindered by the scarcity of material and the fact that in vitro fertilisation (IVF) embryos available for research are usually of poor quality and are, therefore, not representative of normal development. This has led most authors to study individual human embryos, using conventional RT-PCR strategies, which permit analysis of only a few genes. Variability in the expression of genes between individual embryos is characteristic of these studies. In this study, a global RT-PCR strategy has been used, allowing the analysis of an almost infinite number of genes from a single embryo. We have used oocytes, which failed to fertilise and representative pronucleate embryos donated from cycles in which the patient conceived, to investigate possible variability in transcript abundance between individual embryos. We have screened oocytes and embryos for a panel of genes including beta-actin (expressed in 24/28 oocytes, 6/6 pronuclear embryos), the integrins beta1 (17/28 oocytes, 6/6 pronuclear embryos) and beta5 (8/28 oocytes, 5/6 pronuclear embryos), and the apoptotic regulators BCL-2 (20/28 oocytes, 2/6 pronuclear embryos) and BAX (21/28 oocytes, 5/6 pronuclear embryos). The expression of the pro-apoptotic regulator BAX increased in human oocytes following prolonged periods of culture. Overall, patterns of gene transcript presence showed variation between embryos and this was independent of either zona removal or lysis conditions. Pronucleate embryos showed less variation, however, even sibling embryos from the patient did not express an identical subset of genes.