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The aim of this article is to gather 9 thought leaders and their team members to present their ideas about the future of in vitro fertilization and the andrology laboratory. Although we have seen much progress and innovation in the laboratory over the years, there is still much to come, and this article looks at what these leaders think will be important in the future development of technology and processes in the laboratory.

Herein we report the optimization of a series of tricyclic indazoles as selective estrogen receptor degraders (SERD) and antagonists for the treatment of ER+ breast cancer. Structure based design together with systematic investigation of each region of the molecular architecture led to the identification of N-[1-(3-fluoropropyl)azetidin-3-yl]-6-[(6S,8R)-8-methyl-7-(2,2,2-trifluoroethyl)-6,7,8,9-tetrahydro-3H-pyrazolo[4,3-f]isoquinolin-6-yl]pyridin-3-amine (28). This compound was demonstrated to be a highly potent SERD that showed a pharmacological profile comparable to fulvestrant in its ability to degrade ERα in both MCF-7 and CAMA-1 cell lines. A stringent control of lipophilicity ensured that 28 had favorable physicochemical and preclinical pharmacokinetic properties for oral administration. This, combined with demonstration of potent in vivo activity in mouse xenograft models, resulted in progression of this compound, also known as AZD9833, into clinical trials.

In this article, we report our efforts towards improving in vitro human clearance in a series of 5-azaquinazolines through a series of C4 truncations and C2 expansions. Extensive DMPK studies enabled us to tackle high Aldehyde Oxidase (AO) metabolism and unexpected discrepancies in human hepatocyte and liver microsomal intrinsic clearance. Our efforts culminated with the discovery of 5-azaquinazoline 35, which also displayed exquisite selectivity for IRAK4, and showed synergistic in vitro activity against MyD88/CD79 double mutant ABC-DLBCL in combination with the covalent BTK inhibitor acalabrutinib.

STUDY QUESTION Do donor oocyte recipients benefit from preimplantation genetic testing for aneuploidy (PGT-A)? SUMMARY ANSWER PGT-A did not improve the likelihood of live birth for recipients of vitrified donor oocytes, but it did avoid embryo transfer in cycles with no euploid embryos. WHAT IS KNOWN ALREADY Relative to slow freeze, oocyte vitrification has led to increased live birth from cryopreserved oocytes and has led to widespread use of this technology in donor egg IVF programs. However, oocyte cryopreservation has the potential to disrupt the meiotic spindle leading to abnormal segregation of chromosome during meiosis II and ultimately increased aneuploidy in resultant embryos. Therefore, PGT-A might have benefits in vitrified donor egg cycles. In contrast, embryos derived from young donor oocytes are expected to be predominantly euploid, and trophectoderm biopsy may have a negative effect relative to transfer without biopsy. STUDY DESIGN, SIZE, DURATION This is a paired cohort study analyzing donor oocyte-recipient cycles with or without PGT-A performed from 2012 to 2018 at 47 US IVF centers. PARTICIPANTS/MATERIALS, SETTING, METHODS Vitrified donor oocyte cycles were analyzed for live birth as the main outcome measure. Outcomes from donors whose oocytes were used by at least two separate recipient couples, one couple using PGT-A (study group) and one using embryos without PGT-A (control group), were compared. Generalized estimating equation models controlled for confounders and nested for individual donors contributing to both PGT-A and non-PGT-A cohorts, enabling a single donor to serve as her own control. MAIN RESULTS AND THE ROLE OF CHANCE In total, 1291 initiated recipient cycles from 223 donors were analyzed, including 262 cycles with and 1029 without PGT-A. The median aneuploidy rate per recipient was 25%. Forty-three percent of PGT-A cycles had only euploid embryos, whereas only 12.7% of cycles had no euploid embryos. On average 1.09 embryos were transferred in the PGT-A group compared to 1.38 in the group without PGT-A (P LIMITATIONS, REASONS FOR CAUTION Pooled clinical data from 47 IVF clinics introduced PGT-A heterogeneity as genetic testing were performed using different embryology laboratories, PGT-A companies and testing platforms. WIDER IMPLICATIONS OF THE FINDINGS PGT-A testing in donor oocyte-recipient cycles does not improve the chance for live birth nor decrease the risk for miscarriage in the first transfer cycle but does increase cost and time for the patient. Further studies are required to test if our findings can be applied to the young infertility patient population using autologous oocytes. STUDY FUNDING/COMPETING INTEREST(S) No external funding was used for this study. There are no conflicts of interest to declare. TRIAL REGISTRATION NUMBER N/A.

While the treatment of gastrointestinal stromal tumors (GISTs) has been revolutionized by the application of targeted tyrosine kinase inhibitors capable of inhibiting KIT-driven proliferation, diverse mutations to this kinase drive resistance to established therapies. Here we describe the identification of potent pan-KIT mutant kinase inhibitors that can be dosed without being limited by the tolerability issues seen with multitargeted agents. This effort focused on identification and optimization of an existing kinase scaffold through the use of structure-based design. Starting from a series of previously reported phenoxyquinazoline and quinoline based inhibitors of the tyrosine kinase PDGFRα, potency against a diverse panel of mutant KIT driven Ba/F3 cell lines was optimized, with a particular focus on reducing activity against a KDR driven cell model in order to limit the potential for hypertension commonly seen in second and third line GIST therapies. AZD3229 demonstrates potent single digit nM growth inhibition across a broad cell panel, with good margin to KDR-driven effects. Selectivity over KDR can be rationalized predominantly by the interaction of water molecules with the protein and ligand in the active site, and its kinome selectivity is similar to the best of the approved GIST agents. This compound demonstrates excellent cross-species pharmacokinetics, shows strong pharmacodynamic inhibition of target, and is active in several in vivo models of GIST.

How does oocyte cohort size affect IVF treatment outcomes?Retrospective cohort analysis of 10,193 fresh autologous oocyte retrievals among good-prognosis patients35 years from 2009 to 2015. The primary outcome was live birth from a fresh transfer; secondary outcomes included cumulative live birth potential from the retrieved cohort and frequency of severe ovarian hyperstimulation syndrome (OHSS).Live birth per fresh transfer increased as the oocyte cohort increased up to 11-15 oocytes, then plateaued. Beyond 15 oocytes, live birth rates from fresh transfer did not decrease, even at the highest oocyte yields. When accounting for the availability of cryopreserved high-quality supernumerary blastocysts, the cumulative number of potential live births per retrieval continued to increase as oocyte yield increased. Rates of severe OHSS increased rapidly with increasing cohort size above 7-10 oocytes when final oocyte maturation was triggered with human chorionic gonadotrophin (HCG), up to nearly 7% of HCG-triggered retrievals of25 oocytes, but when triggered with gonadotrophin-releasing hormone (GnRH) agonist the severe OHSS rate remained relatively low and stable at approximately 1% even among retrievals of the largest oocyte cohorts.Live birth rates per fresh embryo transfer are highest among cycles with retrieval of 11 or more oocytes. Larger cohorts are not associated with any decline in fresh transfer birth rates. Total potential births per retrieval continue to increase as the number of retrieved oocytes increases. Rates of OHSS remain relatively low after retrieval of large oocyte cohorts if final maturation is triggered with GnRH agonist rather than HCG.

To evaluate factors associated with cryopreserved blastocyst transfer birth outcomes, including age, expansion time, cryopreservation protocol, cryodamage, and number of embryos transferred.Retrospective cohort study.Private infertility practice.Cryopreserved blastocyst transfer patients from January 2003 to April 2012.None.Birth per transfer and children per embryo.Overall live birth per transfer was 32%, with 17% twin births and 0.3% triplets. Live birth per transfer was significantly higher for vitrification compared with slow-freeze (day 5 cryopreservation: 47% vs. 35%; day 6 cryopreservation: 46% vs. 24%), as was live born children per transferred embryo (39% vs. 29% for day 5; 36% vs. 18% for day 6). Birth rates declined only slightly with increasing age at cryopreservation through 37 years, followed by an increasingly rapid decline in success with increasing age thereafter. Live birth rates declined rapidly (49%-18% for vitrification and 37%-10% for slow-freeze) as the percentage of intact cells after cryopreservation decreased from 95%-100% to 70%-79%, with almost no births when the percentage of intact cells was70%. Increasing numbers of embryos per transfer were associated with significant increase in live birth per transfer but significant decrease in children per transferred embryo. Birth rates were much lower for blastocysts with delayed expansion on day 7 (10% per transfer).Birth outcomes from cryopreserved blastocyst transfer are influenced by age, timing of expansion, cryopreservation protocol, visible cryodamage, and the number of embryos transferred. Vitrification substantially improves outcomes versus slow freezing.

Gamete intrafallopian transfer (GIFT) is increasingly accepted as a realistic alternative to in vitro fertilization (IVF), or intrauterine insemination (IUI) for treatment of non-tubal infertility. The lack of information on fertilization capacity of the gametes, the greater cost relative to IUI, and the partly unsubstantiated claims of higher success rates, caused us some concern with the readiness with which GIFT had been accepted as a standard infertility treatment. So we undertook a provisional GIFT programme with these considerations in mind, and we report on the first 91 GIFT cycles performed in our clinic. Sixty of the patients (62 cycles) treated suffered from idiopathic infertility, 12 from minimal endometriosis, 9 from male factor infertility, and 8 from ovulatory dysfunction. An initial clinical pregnancy rate of 41% (38/91) was achieved; pregnancy loss was 23% (9/38), giving a continuing pregnancy rate of 32% (29/91). Given this undeniably encouraging result, and the potential for diagnostic IVF, embryo freezing, and ovum donation with surplus oocytes collected from this GIFT programme, we now have adopted GIFT permanently as a treatment to complement our IVF and IUI programmes.

A conventional view of mammalian fertilization is that the active component of the process: the spermatozoon, by virtue of its progressive motility and acrosomal enzymes, penetrates an otherwise passive oocyte. This concept has placed bias on spermatozoal normality as largely determining the outcome of fertilization; once this has been achieved then the contribution of the spermatozoon is often forgotten, and attention switches to the maternally derived "blue-print" for early embryonic development. Paternal genomic contribution is known to start at the eight-cell stage in the human, but this is usually after the time when early cleavage stage (2 to 8-cell stage) embryos are replaced in human assisted reproductive technologies (ART) procedures such as in vitro fertilization and embryo transfer (IVF-ET). Hence, fundamental abnormal contributions to embryogenesis derived from the fertilizing spermatozoon have often been ignored. Human IVF-ET has permitted far greater powers of analysis of the fertilization event, and fertilization success appears to be determined in such a system by three main factors: spermatozoal quality, oocyte quality, and quality of in vitro culture conditions (the gamete environment). If the second two factors are more carefully controlled than the first, as is the usual emphasis in routine human IVF practice, then any large variation in fertilization rates that are also significantly related to embryonic viability and ultimately pregnancy outcome, may be thought to be more directly associated with original quality of the fertilizing spermatozoon. An analysis of results of 758 IVF cases provides preliminary evidence to show that there is a close association between human in vitro fertilization rate and subsequent embryo viability following replacement. In accepting this hypothesis as a possibility, we should drastically change our attitude from one of the spermatozoon as a robust, simple initiator of embryonic development, and embrace the idea of the vulnerability of such germ cells both during and after their production, and how detrimental influences on this might profoundly affect embryogenesis after successful fertilization.

Eighty-one clinical pregnancies occurred from 508 cycles of intrauterine insemination (IUI) in 242 patients. Following 20 miscarriages 30 pregnancies have been delivered, and 31 remain ongoing at 16 weeks and beyond. This represents a 12% delivered/ongoing pregnancy outcome per cycle of IUI, in women of average age 30.9 years who with their partners had suffered on average 3.65 years of infertility. All forms of non-tubal infertility were treated with pregnancies occurring in all primary categories. No pregnancies occurred in 5 women who underwent IUI in 18 natural cycles. All pregnancies arose in the 237 women who received clomiphene citrate, human menopausal gonadotropin, or a combination of both with ovulation triggered by human chorionic gonadotropin. Dependent upon patient age and duration of infertility, we implement IUI as a frontline treatment for all cases of non-tubal infertility prior to subjecting patients to the more costly and invasive tubal transfer techniques.

Objective To evaluate whether assisted hatching improves clinical outcomes of embryo transfers to good prognosis patients, defined as patients ≤39 years with normal follicle-stimulating hormone (FSH) and E 2 levels, no more than one previous unsuccessful cycle of in vitro fertilization (IVF)–embryo transfer, and good embryo quality. Design Prospective randomized controlled trial. Setting Private assisted reproductive technology (ART) center. Patient(s) One hundred ninety-nine good prognosis patients undergoing IVF–embryo transfer. Intervention(s) In vitro fertilization followed by embryo transfer on day 3 after oocyte retrieval with or without assisted hatching using a 1,480-nm wavelength infrared laser. Main Outcome Measure(s) Clinical intrauterine pregnancy, spontaneous pregnancy loss, and live birth. Result(s) Rates of clinical intrauterine pregnancy with fetal cardiac activity (53% vs. 54% per cycle), spontaneous pregnancy loss (13% vs. 16% per pregnancy), and live birth (47% vs. 46% per cycle) were very similar between treatment cycles with laser-assisted hatching and control cycles in which embryos were transferred without assisted hatching. There were no significant differences between treatment and control groups in any measured clinical outcome parameters. Conclusion(s) Assisted hatching does not improve clinical outcomes among good prognosis patients.

Objective To evaluate a single treatment center's experience with autologous IVF using vitrified and warmed oocytes, including fertilization, embryonic development, pregnancy, and birth outcomes, and to estimate the likelihood of live birth of at least one, two, or three children according to the number of mature oocytes cryopreserved by elective fertility preservation patients. Design Retrospective cohort study. Setting Private practice clinic. Patient(s) Women undergoing autologous IVF treatment using vitrified and warmed oocytes. Indications for oocyte vitrification included elective fertility preservation, desire to limit the number of oocytes inseminated and embryos created, and lack of available sperm on the day of oocyte retrieval. Intervention(s) Oocyte vitrification, warming, and subsequent IVF treatment. Main Outcome Measure(s) Post-warming survival, fertilization, implantation, clinical pregnancy, and live birth rates. Result(s) A total of 1,283 vitrified oocytes were warmed for 128 autologous IVF treatment cycles. Postthaw survival, fertilization, implantation, and birth rates were all comparable for the different oocyte cryopreservation indications; fertilization rates were also comparable to fresh autologous intracytoplasmic sperm injection cycles (70% vs. 72%). Implantation rates per embryo transferred (43% vs. 35%) and clinical pregnancy rates per transfer (57% vs. 44%) were significantly higher with vitrified–warmed compared with fresh oocytes. However, there was no statistically significant difference in live birth/ongoing pregnancy (39% vs. 35%). The overall vitrified–warmed oocyte to live born child efficiency was 6.4%. Conclusion(s) Treatment outcomes using autologous oocyte vitrification and warming are as good as cycles using fresh oocytes. These results are especially reassuring for infertile patients who must cryopreserve oocytes owing to unavailability of sperm or who wish to limit the number of oocytes inseminated. Age-associated estimates of oocyte to live-born child efficiencies are particularly useful in providing more explicit expectations regarding potential births for elective oocyte cryopreservation.

Objective To evaluate implantation of day 5 and day 6 vitrified and slow-frozen blastocysts. Design Retrospective analysis comparing two cryopreservation techniques. Setting Private IVF clinic. Patient(s) Five hundred eight cryopreserved embryo transfer candidates. Intervention(s) Supernumerary day 5 and day 6 blastocysts were vitrified or slow-frozen and transfered after warming or thawing. Main Outcome Measure(s) Comparison of two cryopreservation techniques with respect to survival rate, implantation, and pregnancy rates of day 5 and day 6 blastocysts. Result(s) In 254 vitrified transfer cycles, survival, embryonic implantation, and clinical pregnancy rates for day 5 blastocysts were 95.9%, 33.4%, 48.7%, respectively, and for day 6 blastocysts 97.5%, 25.9%, 42.8%. In 254 slow-frozen transfer cycles, survival, embryonic implantation, and clinical pregnancy rates for day 5 blastocysts were 91.4%, 29.6%, 42.8%, respectively, and for day 6 blastocysts 94.8%, 28.2%, 43.1%. Overall there was a slightly, but not significantly, higher outcome regarding implantation and clinical pregnancy with the use of day 5 blastocysts (31.3% and 45.4%, respectively) versus day 6 blastocysts (26.7, and 42.9%, respectively). Conclusion(s) Vitrification technique yields the same implantation and pregnancy rate as slow-frozen blastocyst transfers. Slow growing embryos can be cryopreserved on day 6, because they yield a satisfactory survival, implantation, and pregnancy rate.

During the last few years, cryopreservation has become a relevant addition to therapeutic concepts in reproductive medicine. New data and publications have made it difficult to maintain an overview of all of the new developments and their results. The focus of interest more recently, especially with the cryopreservation of human oocytes and human ovarian tissue, has been vitrification as an interesting alternative to slow freezing methods. Even though studies investigating the slow freezing of human mature oocytes have resulted in very different survival rates, it could be an option for donor oocyte programs, in the case of threatened ovarian loss or when there is an objection to embryo freezing. An optimal freezing protocol and later use of thawed human ovarian tissue is still a point of discussion. There are encouraging results regarding different kinds of autotransplantation, and recently the first birth after orthotopic autotransplantation of cryopreserved/thawed human ovarian tissue was described in the literature. Independent of any objections to cryopreservation in general, vitrification is a potential and effective alternative to conventional slow cryopreservation, especially for oocytes and embryos. Vitrification might be also be an option for human ovarian tissue; however this is only in its infancy and requires much additional investigation. Our article discusses new trends and results of actual studies regarding these issues.

Embryo cryopreservation has become an ethical necessity due to the way human in vitro fertilization (IVF) infertility therapy has developed. Limited embryonic implantation has by necessity driven IVF therapy to adopt ways to maximize the harvest of oocytes following ovarian hyperstimulation with its attendant risks. Collection of more oocytes has allowed more embryos to be generated to compensate for poor embryonic viability, often leading to transfer of multiple embryos to increase per transfer pregnancy rates. In an era of improving embryonic viability and prevailing trend toward single embryo transfers, production of excessive numbers of surplus embryos appears increasingly inappropriate. At which stage embryo cryopreservation can be undertaken most effectively remains controversial. Embryo cryopreservation nevertheless represents the current solution to the problem of excessive embryo production, but inherently raises ethical concerns for certain couples uncomfortable with what they might perceive to be "experimental" cryostorage, who in extreme circumstances may even choose to limit the number of oocytes inseminated to obviate the production of spare embryos. On a more practical level, cryostored embryos are co-owned by two people who may separate, and as such the embryos then face an uncertain fate, commonly decided in courts of law. Oocyte cryopreservation, if consistent and successful, offers a way to avoid the above complications of routine IVF therapy. Oocytes may need to be cryostored in the event of unforeseen non-production of sperm during IVF therapy, allowing a more measured consideration of donor sperm use or other means of sperm retrieval. Beyond IVF for infertility therapy using a couple's own gametes, oocyte cryopreservation provides a wonderful opportunity to optimize donor oocyte cryo-banking, reducing costs and improving convenience. Meanwhile, frozen embryo donation is an approach that many couples are uncomfortable with, and allows only for retrospective donor screening, and less gametic choice. Advances in ovarian tissue cryopreservation will probably provide the best approach for long term storage of female gametes for women wishing to elect to prolong their reproductive potential, or prior to cancer therapy. However, improved consistency with vitrification technology through standardization of protocols and cell-carrying systems is bringing routine single oocyte cryostorage, at all stages of egg maturity, closer to reality. This, coupled with in vitro maturation, will aid development of oocyte collection protocols using minimal amounts of gonadotropins. All of which will help drive IVF programs to consider cryostorage of excess oocytes and not embryos, inseminating post-thaw/warming only a limited number of oocytes at any one time, in anticipation of the need for only one or two embryos at transfer. The question then is how close are we to being able to provide routine clinical application of human oocyte cryostorage, and when will it be appropriate?

The mitochondrial heteroplasmy present in offspring from IVF and human ooplasm donation is troublesome and merits further exploration in a debate that is already complex and controversial. Improving the understanding of mitochondrial genomics in this context is important because mitochondriopathies can impact crucial cellular processes in renal, cardiovascular, central nervous, and endocrine systems. Relevant epigenetic consequences of mitochondrial heteroplasmy include associated abnormalities in mitochondrial translation products. Furthermore, as transmission and inheritance patterns of mtDNA are species-specific, it remains to be proven if findings derived from animal studies are applicable to human offspring. As an alternative to gamete research and proteomics based on animal experimentation, continued molecular characterization of the de novo human mitochondriopathies is posed to offer further insights regarding mitochondrial heteroplasmy. In this context, because knowledge of human mitochondrial genetics remains limited and the risks associated with ooplasm donation cannot be quantified, we do not favor its use for our patients at present. However, the small number of infants already conceived from this experimental approach warrant careful longitudinal evaluation. In particular, observational study of the few children born after ooplasm donation could provide opportunities to assess human mtDNA transmission and inheritance. Such findings could help identify features distinguishing natural mtDNA heteroplasmy from heteroplasmy observed after ooplasm donation. Future investigations should also quantify the degree any such heteroplasmy can exist innocuously. Disclosure of mtDNA mutations potentially affecting children conceived from IVF and ooplasm donation must be included during patient education at centers contemplating such treatment.

The target of any cryopreservation procedure should be to ensure high survival rates of living cells after thawing. Two important parameters determine the success of any cryopreservation protocol: the manner in which cells regain equilibrium in response to cooling, and the speed of freezing (cooling rate). Slow-rate freezing protocols result in the formation of ice crystals during cooling and warming. Vitrification, in which high cooling rates in combination with a high concentration of cryoprotectant are used, does not produce any ice crystals during cooling and warming. However, there is a practical limit to the attainable cooling speed, and also a biological limit to the concentration of cryoprotectant tolerated by the cells during vitrification. Although post-warming survival depends on the species, the developmental stage and the quality of the embryos being vitrified, it seems clear that vitrification methods are increasingly successful and might be a better method than slow cooling procedures in the field of cryobiology. Many of the potential problems and benefits underlying vitrification as a method of choice for embryo cryopreservation in clinical embryology will be discussed in this review.

A 39 year-old woman with previous salpingectomy developed a symptomatic heterotopic right cornual pregnancy identified by transvaginal ultrasonography at six weeks' gestation. The patient had previously undergone an ipsilateral partial salpingectomy, and the conception was established four months later after one cycle of controlled ovarian hyperstimulation, in vitro fertilization (IVF) and embryo transfer. We performed immediate surgical excision of the ectopic implantation with conservation of the intrauterine pregnancy. Progesterone was administered as 200 mg/d lozenge (troche) plus 200 mg/d rectal suppository, maintained from day of embryo transfer through the perioperative period and until 11th gestational week. Following an uneventful obstetrical course, a healthy male infant was delivered by cesarean at term. In this report, we review the incidence and significance of heterotopic gestation in the context of IVF/embryo transfer. Risk factors for complex intra- and extra-uterine pregnancies are also outlined. Additionally, the clinical management of heterotopic pregnancy, including a novel approach to progesterone supplementation, is discussed.

Human embryos not chosen for fresh transfer or cryopreservation were maintained in extended in vitro culture for up to 9 days after fertilization to observe blastocyst formation and hatching features. These non-transferred embryos were derived from 64 consecutive IVF cycles, and were not cryopreserved either because of compromised morphology or because the patients did not consent to cryopreservation for personal reasons. Embryos were cultured individually to monitor daily growth until developmental arrest, and differential blastocyst formation and hatching were analysed among groups of patients and embryos. In the population studied, hatching occurred most commonly on day 7 after fertilization (range 5-9 days). A total of 301 blastocysts was observed, of which 116 (38.5%) eventually hatched in vitro irrespective of day of formation. A trend towards earlier blastocyst formation and a greater likelihood of hatching was noted in this population. Both blastocyst formation and hatching appeared negatively correlated with increasing maternal age and higher basal serum FSH concentrations on day 3 of development, although these trends did not reach statistical significance. Comparison of intracytoplasmic sperm injection (ICSI) (n = 25) and conventional insemination (n = 39) cycles showed a similar rate of blastocyst formation in both groups (54 and 52%, respectively; P0.05), but hatching patterns varied significantly between these groups (4.1 versus 61.6%, respectively; P0.0001). The discovery of marked impairment of hatching among non-transferred ICSI embryos supports the case for reconsideration of the appropriateness of assisted blastocyst hatching in selected cases.

To compare standard density gradient centrifugation sperm preparation with a novel non-centrifugation-based dual-chamber capillary dish in efficiency for motile human sperm separation, approximately 3 mL fresh ejaculate specimens was obtained from 21 men (median age = 32 years. range 26-42 years) undergoing infertility evaluation. For each specimen, half of the sample was processed with a standard 45%/90% density gradient preparation (PureSperm. Nidacon International, Gothenburg, Sweden) followed by semen analysis. The other half was incubated in the Zech glass capillary dish (Astromedtec, Salzburg, Austria) consisting of 2 concentric wells overlaid by a U-ring and coverglass. After approximately 3 h, a 1-mL sample was taken from the central chamber and analyzed. Percentage motile sperm recovery, absolute (motile) cell number, and path velocities were compared for spermatozoa obtained from both methods. Both techniques reduced overall sperm concentration while enriching specimens with more motile spermatozoa. A trend towards higher % recovery of motile spermatozoa (p = .264) was observed with the Zech device, but at a cost of fewer absolute numbers of higher velocity cells (p = .004). The Zech device, therefore, localized a very small population of motile sperm without exposure to centrifugation stress, which has been considered potentially harmful to spermatozoa. This technique could theoretically improve efficiency by reducing time required to identify motile cells in in vitro fertilization where intracytoplasmic sperm injection is planned. However, refinements in incubation interval and suspension volumes are needed before this technique can be considered comparable to the density gradient method in recovering sperm for use in intrauterine insemination.

Objective: To measure plasma homocysteine, androgen, and insulin concentrations in women with normal and polycystic-appearing ovaries in an infertility setting. Methods: Among women referred for infertility evaluation (n = 54), homocysteine, androstenedione, DHEAS, total testosterone, fasting insulin/glucose and methyltetrahydrofolate reductase (MTHFR) polymorphism status (C677T mutation) were studied. Ovaries were examined via transvaginal sonogram by one observer and scored as either normal (n = 18) or polycystic (n = 36). Results: When polycystic ovaries were identified, mean total testosterone was significantly higher than when non-polycystic ovaries were present (p = 0.01), although no measured androgen was outside the normal reference range in either group. Average BMI was higher in the polycystic group, but the difference was not significant (p = 0.10). We observed a trend toward higher mean fasting insulin levels in women with polycystic ovaries, but this increase did not reach statistical significance (p = 0.07). Median plasma homocysteine was identical (7.0 mmol/l) in both populations, and no study subject exceeded the current recommended maximum reference value. Conclusions: In this population, the presence of polycystic ovaries was associated with higher serum androgens (especially total testosterone) although none of the measured androgens were above the normal range. While fasting insulin levels were also higher in this group, median plasma homocysteine levels were similar irrespective of ovarian morphology. Concomitant plasma homocysteine derangements in this population of young, lean patients with polycystic-appearing ovaries seem unlikely. Further studies are needed to clarify the role(s) of homocysteine in human reproductive physiology.

usromosomal abnormalities are responsible for a great deal of embryo wastage, which is reflected, at least partially, in decreased implantation and increased miscarriage in older women. To address this problem the transfer of only chromosomally normal embryos previously selected by preimplantation genetic diagnosis (PGD) has been proposed. We designed a multi-centre in-vitro fertilization (IVF) study to compare controls and a test group that underwent embryo biopsy and PGD for aneuploidy. Patients were matched retrospectively, but blindly, for average maternal age, number of previous IVF cycles, duration of stimulation, oestradiol concentrations on day +1, and average mature follicles. All these parameters were similar in test and control groups. Only embryos classified as normal for those chromosomes were transferred after PGD. The results showed that the rates of fetal heart beat (FHB)/embryo transferred between the control and test groups were similar. However, spontaneous abortions, measured as FHB aborted/FHB detected, decreased after PGD (P0.05), and ongoing pregnancies and delivered babies increased (P0.05) in the PGD group of patients. Two conclusions were obtained: (i) PGD of aneuploidy reduced embryo loss after implantation; (ii) implantation rates were not significantly improved, but the proportion of ongoing and delivered babies was increased.

Co-culture techniques using fetal bovine uterine fibroblasts or bovine oviductal epithelial cells have improved embryonic development prior to replacement in humans. In initial co-culture trials, embryo development and implantation rates increased after just 1 day in culture. The most overt characteristics noted following co-culture were improved blastomere development and characteristics, reduced fragmentation, and the appearance of swollen blastomeres. In addition, an increase in the incidence of zona thickness variation was detected. Improved development of polyspermic and supernumerary embryos to the blastocyst stage was noted in initial trials. Retrospective analysis indicated that certain patient subgroups benefit the most from co-culture. As a result, co-culture is now applied routinely to patients that have previously failed attempts at in-vitro fertilization (IVF) and/or have endocrine imbalances such as polycystic ovarian syndrome and elevated day 3 concentrations of follicle stimulating hormone (FSH). The use of co-culture prior to or following cryopreservation has also proven to be beneficial to human embryos. The proposed beneficial mechanisms thought to improve embryonic development include a secretory and/or a scavenging role. Evidence describing the postulated benefits is discussed.

Since the first successful application of micromanipulationin human in-vitro fertilization 10 years ago, the breadth of its application has expanded to encompass single sperm injection, zona ablation, polar body and blastomere biopsy, and very recently the first reports of both cytoplasmic and nuclear transfer.

Objective: To analyze the efficacy of intracytoplasmic sperm injection (ICSI) for "rescue" of failed conventional insemination cycles. Design: Retrospective clinical study. Setting: Private infertility clinic. Patient(s): Fifty-four couples with non-male factor infertility undergoing routine IVF within our clinic. Intervention(s): Twenty- to 24-hour-old unfertilized mature oocytes from conventional IVF cycles in which fertilization failure was complete were reinseminated by ICSI to attempt late fertilization and rescue of the otherwise failed IVF cycle. Main Outcome Measure(s): Late fertilization, pregnancy, and embryonic implantation. Result(s): Intracytoplasmic sperm injection reinsemination of 489 unfertilized mature oocytes caused degeneration in 50 (10.2%) oocytes, gave rise to normal fertilization in 215 (44.0%) of the injected oocytes, and allowed ET in 48 of the 54 cycles in which initial complete fertilization failure had occurred. One hundred sixty-four (76.3%) of 215 late-fertilized oocytes either were used for fresh transfer or were frozen-stored. Eight viable pregnancies resulted, yielding a 14.8% pregnancy rate per initiated cycle. Conclusion(s): Barring potential concerns regarding the chromosomal normality of embryos arising from reinsemination, our results suggest that ICSI is a relatively successful means of rescuing conventional IVF cycles in which fertilization fails completely.

Mosaicism was studied in good quality embryos from four different centres in order to assess the effects of follicular induction and exposure to laboratory conditions on chromosomal status. The donated embryos were fully biopsied and analysed by fluorescence in-situ hybridization using probes for chromosomes X, Y, 13, 18 and 21, simultaneously. The number of abnormal cells present indicated the division at which mosaicism first occurred (4/4 cells at first division, 2/4 cells at second, 2/8 at third). The rate of mosaicism in embryos from different centres varied greatly (P < 0.001). Most of the mosaic embryos were obtained before 1991. In one clinic increased mosaicism was found in embryos obtained before 1991 when compared to embryos obtained thereafter. The results suggest that certain culture conditions and/or hormonal stimulation protocols may induce chromosomal abnormalities and partly explain differences in pregnancy rates between in-vitro fertilization centres.

In two separate prospectively randomized trials, intracytoplasmic sperm injection (ICSI) cycles were studied in a controlled manner to monitor the effects of either bovine oviductal epithelial cell co-culture (n = 119) or assisted hatching by zona drilling (n = 100). In the first study, immediately following ICSI, all eggs were placed directly either onto partial monolayers of bovine oviductal cells or into regular culture medium. Although the embryo developmental rate was apparently compromised in part by the presence of the co-culture cells, ultimately there were no significant differences in either the viable pregnancy rate (31.6% co-culture versus 29.0% control) or the embryonic implantation rate (11.4% co-culture versus 13.6% control). Assisted hatching also had no significant impact on ICSI cycle outcome in terms of either the viable pregnancy rate (30.0% assisted hatching versus 32.0% control) or the embryonic implantation rate (8.5% assisted hatching versus 13.5% control). However, in female patients aged > or = 35 years, assisted hatching appeared to convey a marginally significant benefit in terms of both the viable pregnancy rate (35.5% assisted hatching versus 11.1% control) and the embryonic implantation rate (10.3% assisted hatching versus 3.1% control). It seems that the overall improvement of ICSI cycle outcome cannot be achieved by the general application of either co-culture or assisted hatching. Nevertheless, it is possible that there remain specific patient groups that might benefit from selected use of either of these modalities.

Purpose : This retrospective analysis was designed to assess the performance of human embryos following cryopreservation based on whether they were originally developed in standard culture medium (65 cycles, 223 embryos) or cocultured on partial monolayers of bovine oviductal epithelial cells (63 cycles, 198 embryos). Embryo cryosurvival and implantation were compared between the study group and the contemporaneously matched controls. Results : During a 2-year period when no factors of the cryopreservation program were altered, 63 transfers of 159 surviving thawed control cleavage-stage embryos (71.3% survival) that were 54% intact gave rise to 11 viable pregnancies (17.5%/ET), to yield an implantation rate of 6.9% per embryo. Sixty-three transfers of 147 thawed cocultured embryos (74.2% survival) that were 61% intact gave rise to 17 viable pregnancies (27%/ET), which gave an implantation rate of 13.6% per embryo that was significantly higher than the control group (P < 0.05). Conclusion : Coculture of embryos prior to cryopreservation does not appear to improve cryosurvival ; however, it does improve implantation postthaw compared with embryos following standard culture prior to cryopreservation.

Human embryo cryopreservation is now a fully established adjunct to assisted reproduction, with thawed embryos implanting at a rate approaching that of fresh embryos. As with fresh embryos, the quality of frozen-thawed embryos is affected by patient age at the time of oocyte retrieval, and by culture conditions. Ovarian stimulation incorporating gonadotropin-releasing hormone analogs does not appear to be detrimental to cryopreservation success, and natural or artificial thaw cycles seem equally effective. Successful cryopreservation seems feasible for one- to eight-cell embryos and blastocysts using a variety of protocols. Selection criteria for embryos to be frozen can vary, and embryo quality has the most significant impact on post-cryopreservation viability. Unfertilized oocyte cryopreservation is coming closer to routine clinical application.

A rare case of serous cystadenofibroma of the fallopian tube was discovered during evaluation for in vitro fertilization-embryo transfer. Bilateral tubal occlusion was noted on hysterosalpingogram, and a right adnexal cyst, initially thought to be of ovarian origin, was identified by office transvaginal sonography. Laparoscopy revealed a 5.5-cm, fluid-filled mass involving the distal aspect of the right fallopian tube. Both ovaries and uterine exterior appeared grossly normal. The cyst was decompressed and removed intact without incident through a 5-mm laparoscopic cannula. The mass showed histologic features consistent with benign serous cystadenofibroma. The patient had an uncomplicated postoperative convalescence and continued to do well 3 months after surgery.

Older patients and those who consistently return for embryo transfer but without implantation were studied to see if a combination of day 3 assisted hatching and co-culture (AH+CC) might be beneficial compared to assisted hatching alone (AH-alone). Female patients of > or = 38 years and couples who had previously failed to implant embryos three times or more were prospectively and randomly assigned to either an experimental or a control group. In the experimental group all embryos were co-cultured on partial monolayers of bovine oviductal epithelial cells for 2 days followed by assisted hatching by zona drilling (AH+CC). All control embryos were cultured by standard procedures until day 3 when they also underwent zona drilling prior to uterine transfer (AH-alone). With 50 cycles in each group there was unfortunately a marginal bias against the AH+CC group in that these patients had undergone a higher number of previous transfer cycles. There was a marginally lower percentage of fragmentation and a significantly higher degree of zona thickness variability in the AH+CC embryo group. Embryonic implantation was significantly increased (P < 0.05) in the AH+CC group (18%) when compared to the AH-alone group (10%). This difference was reflected in a significantly higher (P < 0.05) initial pregnancy rate (52 versus 32%) in the AH+CC group, and a higher (not significant) viable pregnancy rate (38 versus 22%).

This retrospective, descriptive study was designed to determine the effectiveness of using clomiphene citrate in relatively high daily doses (100, 150, and 200 mg) with simplified monitoring for in vitro fertilization in a private office and surgical center. The self-selected study population comprised 109 women who were 25-42 years old, including 26 women whose husbands had mild male-factor infertility.During January 1992 through December 1993, 165 stimulation cycles resulted in 137 egg retrievals, 24 clinical pregnancies (17.5%), and 20 viable pregnancies (14.5%). Cycles that could not be completed (28/165 or 16.9% of all cycles) involved luteinizing hormone surges, insufficient follicles, or low estradiol levels. There were no pregnancies in patients who were 40 years or older or who received 200 mg daily doses of clomiphene citrate. The viable pregnancy rate among patients with male factor infertility was 7.7% (2/26). For non-male-factor infertility patients who were younger than 40, the viable pregnancy rate was 17.6% (18/102).The simplified monitoring method did not appear to compromise the results.

Objective To describe the convergence of five rare phenotypic features in a woman with premature ovarian failure referred for reproductive endocrinology evaluation. Design Case report and literature review. Setting Major urban infertility referral center. Patient(s) A 24-year-old nulligravida with cutis marmorata telangiectatica congenita (CMTC), premature ovarian failure, unilateral ovarian agenesis, septate uterus, and de novo balanced autosomal translocation. Intervention(s) High-resolution chromosomal evaluation, radiographic study of reproductive organs, and assessment of endogenous estrogen production. Main outcome measure(s) Patient counseling regarding future reproductive options (i.e., donor oocyte in vitro fertilization/embryo transfer), and satisfactory management of hypoestrogenism using oral contraceptives. Result(s) We identified a balanced reciprocal translocation 46,XX t(8;9)(q22.1;p24.1), and confirmed unilateral ovarian agenesis with midline intrauterine septum. Conclusion(s) Although genetic factors considered contributory to premature ovarian failure usually involve the X chromosome, in our patient a previously undescribed autosomal translocation was identified in association with CMTC, a rare vascular disorder. The fundamental role of follicular oxygenation in oocyte competence and subsequent ovarian function is discussed. From the clinical and laboratory findings evident in this unusual case, a developmental hypothesis connecting the vascular abnormalities of CMTC and premature ovarian failure is offered.

Objective: To report a normal twin delivery after transfer of two fresh day 7 blastocysts. Design: Case report. Setting: Private infertility clinic. Patient(s): A 35-year-old woman with a 6-year history of primary infertility with significant pelvic adhesions. Intervention(s): Review of individual IVF-ET therapy cycle. Main Outcome Measure(s): Full-term delivery after day 7 blastocyst transfer. Result(s): During the patient's first IVF-ET cycle, the decision was made to undertake blastocyst transfer after extended culture. No blastocysts had formed until late on day 6, by which time the patient had been hospitalized with a renal stone. Subsequently, on day 7, the patient was asymptomatic and presented for embryo transfer, and after assisted hatching, two expanded blastocysts were transferred to her uterus under ultrasound guidance. After confirmation of implantation of a viable twin, pregnancy was uneventful with no obstetrical complications, and a dizygotic twin was delivered vaginally at 38 weeks of gestation. Conclusion(s): Few reports have been made regarding viability of more slowly developing blastocysts; however, this case indicates that blastocysts that did not fully expand until day 7 of extended in vitro culture are still able to implant after superovulation and IVF-ET therapy. Assisted hatching of these embryos may have been beneficial in achieving this successful outcome by hastening the blastocyst hatching, allowing more rapid contact with the endometrium.

Two hundred eighteen consenting patients entered a randomized study of the application of chemical zona pellucida thinning on their day 3 embryos, prior to uterine transfer. Of those control patients (n = 108), whose embryos remained unmanipulated, 40 (37.0%) have ongoing/delivered pregnancies, while in the experimental group (n = 110), whose embryos had their zonae pellucidae chemically thinned, there are 49 patients (44.6%) who have ongoing/delivered pregnancies. Although this difference is not significant, clearly the application of this micromanipulative intervention has not been detrimental, and this bodes well for routine application of embryonic micromanipulation procedures in general. Certain patient subgroups were studied including older women, those with elevated basal follicle stimulating hormone levels, patients with embryos of differing zona thickness, and patients with embryos of differing uniformity of zona thickness. No significant influence of chemical removal of the outside of the zona on the implantation rate of embryos in any of these subgroups was observed other than a marginally significant (P = 0.095) improvement of implantation of embryos with less than 4.0 microns variation in zona thickness when chemical zona thinning was applied. Failure of chemical zona thinning to enhance human embryo implantation significantly, compared to assisted hatching by complete zona drilling, strongly suggests that the bilayered human zona pellucida needs to be fully breached, unlike that of the mouse.

Etanercept (Enbrel; Wyeth-Ayerst/Immunex Inc, Seattle, WA, USA) is a subcutaneously administered novel fusion protein consisting of the extracellular ligand-binding domain of the 75 kD receptor for tumor necrosis factor-alpha (anti-TNFalpha) and the Fc portion of human IgG1. The agent is synthesized by plasmid transfection of a Chinese hamster ovary cell line, utilizing recombinant DNA technology. Etanercept was approved by the US FDA for treatment of multi-drug resistant rheumatoid arthritis in 1998, but no human data exist regarding the impact of anti-TNFalpha therapy on human reproductive function or its use before ovulation induction. As TNFalpha potentiates collagenolysis via matrix metalloproteinase gene expression (thereby facilitating ovulation), there exists a theoretical risk that TNFalpha-inhibition could exert an undesirable effect on ovulation and pregnancy. In this report, we describe the first case of ovulation induction, intrauterine insemination, normal pregnancy and singleton delivery of a healthy infant following chronic (1 year) pre-ovulatory TNFalpha-inhibitor therapy for rheumatoid arthritis. Reproductive endocrinologists and obstetrician-gynecologists should be familiar with etanercept therapy in the context of severe rheumatic disease, and offer appropriate reassurance regarding its safe use for infertility patients planning ovulation induction.

The rate of successful implantation after replacement of frozen-thawed embryos in in vitro fertilization is commonly only 5% to 10% per embryo. A limiting factor may be inability of otherwise viable embryos to be released from the intact zonae pellucidae. Culture conditions and/or cryopreservation in in vitro fertilization may affect the zona and impair blastocyst hatching. Therefore opening of the zona by partial slicing by means of micromanipulation before replacement of early cleaved embryos may improve chances of eventual hatching (referred to as assisted hatching). In 65 thawed embryo replacement cycles methyl-prednisolone and antibiotics were given for 4 days mid cycle. Assisted hatching was performed in 32 cycles, with 33 cycles left as controls. Patient's age, infertility, cycle supplementation, and number of thawed and replaced embryos did not differ significantly between the two groups. Rates of viable embryonic implantation were 16% (10/63) and 9% (6/64) in the assisted hatching and control groups, respectively. Group sizes need approximately to double before this trend toward improved implantation with the use of assisted hatching reaches statistical significance.

Preimplantation genetic diagnosis (PGD) identifies genetic abnormalities in preimplantation embryos prior to. embryo transfer. PGD is an exciting technology that may improve the likelihood of a successful pregnancy and birth for five distinct patient groups: (1) those with infertility related to recurrent miscarriages or unsuccessful in vitro fertilization (IVF) cycles, (2) those with unexplained infertility, (3) advanced maternal age, (4) severe male factor infertility, and (5) couples at risk for transmitting a hereditary disease to their offspring. PGD is always performed following an IVF cycle where multiple oocytes are retrieved and fertilized. Sophisticated techniques such as multiprobe, multicolor fluorescence in situ hybridization are used to test single cells for structural or numerical chromosome abnormalities, whereas the polymerase chain reaction, linkage analysis, and DNA sequencing are used to analyze single cells for disease-specific DNA mutations. PGD allows one to transfer only those embryos identified as being free of genetic abnormalities, thus potentially increasing the implantation rate and decreasing the miscarriage rate. These technologies identify embryos free of specific genetic abnormalities and may increase the likelihood of achieving the patient's goal: the birth of a healthy infant.

Objective To evaluate viability and implantation potential of cryopreserved blastocysts according to the day of blastocyst expansion and cryopreservation. Design Retrospective study. Setting Private ART center. Patient(s) Three hundred and seventy-five patients undergoing embryo transfer with cryopreserved blastocysts. Intervention(s) Blastocyst cryopreservation on day 5, 6, or 7 after oocyte retrieval according to the day of blastocyst expansion and subsequent embryo transfer. Main Outcome Measure(s) Clinical pregnancy rate (PR) per embryo transfer. Result(s) Clinical PRs were similar between blastocysts cryopreserved on day 5 and blastocysts cryopreserved on day 6 (32% vs. 28%). The clinical PR was lower for blastocysts cryopreserved on day 7 (15%), but this difference was not statistically significant after accounting for the number of embryos per transfer ( P =.15). Conclusion(s) Viability and implantation potential are similar for day 5 and day 6 blastocyst cryopreservation. Viability may be reduced for blastocysts cryopreserved on day 7, but not to the extent suggested by reports of fresh transfers. These results suggest that reduced success rates associated with fresh transfers of later developing blastocysts may be the result of asynchrony with endometrial receptivity instead of poorer embryo quality.

This paper aims to describe the relation between standard semen analysis parameters (concentration, motility and morphology) and sperm chromatin structure assay (SCSA) results among patients referred for infertility evaluation. Healthy males (n=136) seeking infertility consultation were evaluated prospectively by semen analysis and sperm chromatin structure assay (SCSA). Significant inverse correlations were observed between high sperm concentration and DNA fragmentation index (DFI) and high DNA stainability (HDS) (r=- 0.45; P0.001, and r=- 0.40; P0.001, respectively). Both progressive motility and normal morphology were also strongly inversely correlated with DFI and HDS. However, in stratified analysis the correlation between concentrationor =20 M/ml and DFI, and concentrationor =20 M/ml and HDS were not significant (P=0.31 and 0.38, respectively). For men with sperm motilityor =40% the correlation between motility and HDS was not significant (P=0.22), but between motility and DFI the correlation remained significant (P=0.04). Although strong correlations between DFI, HDS and semen analysis findings were noted in the overall study population, when oligozoospermic and asthenozoospermic patients were analysed separately the correlation between concentration and sperm chromatin fragmentation was not significant. For such men, SCSA appears to be a diagnostic variable independent of the semen analysis, providing information about nuclear abnormalities not readily apparent from standard semen analysis alone. Additionally, SCSA data may offer explanations for previous miscarriage, providing closure for some couples contemplating future use of anonymous donor sperm.

Objective To perform preimplantation genetic diagnosis (PGD) with 1.48-μm infrared diode laser assistance during embryo biopsy for two patients undergoing IVF. Design Case reports. Setting Private ART laboratory. Patient(s) Two couples undergoing IVF for infertility therapy, both of whom had previously delivered offspring afflicted with spinal muscular atrophy (type 1) after IVF therapy, and who underwent subsequent cycles of IVF coupled with PGD to screen for this disorder. Intervention(s) Two individual IVF cases involving intracytoplasmic sperm injection (ICSI), embryo biopsy with laser assistance, and PGD. Main outcome measure(s) The ease and apparent safety of human embryo biopsy using a 1.48-μm infrared laser for partial zona pellucida (ZP) dissection to assist with embryo blastomere biopsy was evaluated. Result(s) Both couples were deemed to have some unafflicted embryos for transfer on the fifth day of development after blastomere biopsy in conjunction with PGD. Patient A had a singleton pregnancy and delivered a healthy normal singleton male. Patient B had a twin pregnancy; however, one twin was spontaneously lost at 10 weeks but she ultimately delivered a healthy normal singleton male. Conclusion(s) These successful outcomes help to demonstrate the efficacy and safety of laser-assisted embryo biopsy to facilitate PGD screening.

To describe the reproductive outcome following intracytoplasmic sperm injection (ICSI) for male factor infertility associated with Crohn's disease and 6-mercaptopurine (6-MP) chemotherapy.The male partner of a couple suffered from severe Crohn's disease and received a 3-month course of 6-MP for this condition. Two spontaneous conceptions were established before 6-MP, although post-chemotherapy semen analysis found the sperm concentration to be 8,000/mL. In vitro fertilization (IVF) with ICSI and embryo transfer was performed.The woman underwent an uncomplicated controlled ovarian hyperstimulation sequence using a combined rec-FSH+hMG protocol, following late luteal phase pituitary downregulation. This culminated in the retrieval of 18 oocytes, 11 of which were fertilized with ICSI. She later delivered a normal male infant without urogenital anomaly. Four nontransferred blastocysts were cryopreserved.This report describes the first successful birth after ICSI for severe oligozoospermia associated with Crohn's disease and 6-MP therapy. We outline salient features of Crohn's disease, 6-MP pharmacology, and their relevance to human fertility.

We report the delivery of healthy monozygous (MZ) twins in a 31 year-old nulligravida following gonadotrophin ovulation induction, ICSI, assisted hatching and fresh embryo transfer. Although a sonogram on day 35 confirmed that two of four transferred embryos had implanted, a second transvaginal sonogram 1 week later showed each gestational sac had two conceptuses-all four were associated with distinct amniotic compartments. Cardiac activity was observed in all four embryos. At 12 weeks and 5 days gestation, chorionic villus sampling was performed on fetuses 1 and 2 which were euploid for chromosomes 13, 18, 21, X and Y via fluorescence in-situ hybridization analysis. Subsequent KCl injection into sacs 3 and 4 resulted in asystole for these fetuses, while cardiac activity in sacs 1 and 2 remained unchanged after reduction. A twin vaginal delivery occurred at 36 weeks gestation, resulting in the birth of two male infants and one placenta. This case represents the first known report of human quadruplet pregnancy consisting of two MZ twin sets conceived by assisted reproductive techniques. Our report reviews proposed mechanisms for explaining twinning, with special emphasis on zona pellucida micromanipulation and subsequent MZ twin induction.

Objective To report the first conception and delivery following transfer of thawed human blastocysts maintained in extended in vitro culture with cryopreservation at day 6 and 7. Design Case report. Setting Major urban infertility referral center. Patient(s) A 26-year-old woman with pelvic endometriosis and two prior unsuccessful in vitro fertilization/embryo transfer (IVF-ET) attempts. Intervention(s) The patient underwent controlled ovarian hyperstimulation using a combined FSH + hMG protocol, and 24 oocytes were retrieved. Main outcome measure(s) Dizygotic twin delivery after IVF and intracytoplasmic sperm injection (ICSI), assisted embryo hatching, and ultrasound-guided transfer of cryopreserved blastocysts. Result(s) After three embryos were subjected to assisted hatching, they were transferred fresh on day 3, but no implantation occurred. All nontransferred embryos (n = 11) were observed during extended in vitro culture and three blastocysts were selected for cryopreservation on day 6 and 7; thaw and transfer occurred the following month and a pregnancy was achieved. Dizygotic twins (female/female) were delivered by cesarean in the early third trimester. Conclusion(s) Substantial advancements have been made in the field of embryo cryogenics and in vitro fertilization, but controversy remains regarding the value of freezing late-developing human blastocysts. Here we describe the first reported live births with IVF after extended in vitro culture and cryopreservation at day 6 and 7 after fertilization.

The purpose of this study was to demonstrate the safety and efficacy of vitrification of human pronuclear stage (PN) embryos in the human assisted reproduction laboratory. Using single pronucleate (1PN) and three pronucleate (3PN) zygotes, the impact of vitrification in the Flexipet denuding pipette (FDP) as a carrier was assessed in terms of survival, embryonic development and blastocyst formation when compared according to the PN number, and unvitrified controls. A total of 65 1PN and 152 3PN zygotes were vitrified; after warming 82% (53/65) of 1PN and 90% (137/152) of 3PN survived. The overall percentage of warmed zygotes (1PN and 3PN) that cleaved and reached 2-cell stage did not differ (χ 2 ; P = 0.32) from the control groups (77%; 147/190 versus 85%; 115/136). In addition, when the cleavage behaviour was examined on day 3 for ≥4-cell stage, no significant differences (χ 2 ; P = 0.95) were observed between the vitrified group and the unvitrified control groups (74%; 109/147 versus 77%; 89/115). Comparing the developmental potential up to cavitation and blastocyst formation on day 5, the overall outcome of the vitrified PN was 31% compared with 33% for the controls (χ 2 ; P = 0.76). The simple vitrification protocol used in this study, and these data highlight the usefulness of vitrification using FDP as a consistent and effective cryopreservation method for pronuclear zygotes, and a suitable alternative to slow cryopreservation protocols.

As early as 1985, ice-free cryopreservation of mouse embryos at -196 degrees C by vitrification was reported in an attempted alternative approach to cryostorage. Since then, vitrification techniques have entered more and more the mainstream of animal reproduction as an alternative cryopreservation method to traditional slow-cooling/rapid-thaw protocols. In addition, the last few years have seen a significant resurgence of interest in the potential benefits of vitrification protocols and techniques in human-assisted reproductive technologies. The radical strategy of vitrification results in the total elimination of ice crystal formation, both within the cells being vitrified (intracellular) and in the surrounding solution (extracellular). The protocols for vitrification are very simple. They allow cells and tissue to be placed directly into the cryoprotectant and then plunged directly into liquid nitrogen. To date, however, vitrification as a cryopreservation method has had very little practical impact on human-assisted reproduction, and human preimplantation embryo vitrification is still considered to be largely experimental. Besides the inconsistent survival rates that have been reported, another problem is the wide variety of different carriers and vessels that have been used for vitrification. Second, many different vitrification solutions have been formulated, which has not helped to focus efforts on perfecting a single approach. On the other hand, the reports of successfully completed pregnancies following vitrification at all preimplantation stages is encouraging for further research and clinical implementation. Clearly, however, attention needs to be paid to the inconsistent survival rates following vitrification.

The complete androgen insensitivity syndrome (AIS) is a sub-type of X-linked male pseudohermaphroditism resulting from total dysfunction of the androgen receptor. Affected patients are phenotypically female despite a 46,XY genotype; gonadal tissue displays a classic Sertoli cell-only pattern on microscopic examination. We describe the diagnosis and management of a 19(1/2)-year-old patient who presented for primary amenorrhea and absent cervix, identified incidentally during a routine Pap test. Serum total testosterone was elevated (725 ng/dl) and the karyotype was 46,XY. Molecular investigation for specific gene defect(s) causing disruption and functional incapacity of the androgen receptor was undertaken for the proband and her only sibling. From this we discovered a previously unknown hemizygous mutation (A--T) in exon 4 of the androgen receptor gene, associated with replacement of asparagine (AAT) with tyrosine (TAT) in the resultant androgen receptor protein [N705Y]. Bidirectional, non-isotopic sequence analysis of exon 4 was next undertaken for the proband's sister who was found to be heterozygous for this mutation. Psychological and genetic counseling was provided to both individuals; the patient underwent an outpatient laparoscopic orchiectomy without complication. She continues to receive oral hormone replacement therapy following an oral contraceptive model. In this report, the clinical approach to AIS is outlined from a reproductive endocrinology perspective with special emphasis on psychological counseling and laboratory methods employed to confirm the diagnosis at the molecular level. We also outline other recently described mutations of the androgen receptor gene (Xq11-12) which have been associated with AIS.

To describe serum levels of human chorionic gonadotropin (hCG) as a function of hCG injection method (subcutaneous vs. intramuscular) among infertile women undergoing ovulation induction.Prospective, randomized clinical trial.Major urban infertility referral center.Women presenting for infertility evaluation and ovulation induction.Controlled ovarian hyperstimulation was followed by 5,000 IU urinary (nonrecombinant) hCG injection, given intramuscularly (i.m.) or subcutaneously (s.c.).Serum hCG levels measured 24 hours after administration of hCG, and patient tolerability of injected hCG.There were no statistically significant differences in age or body mass index (BMI) among patients receiving hCG s.c. (n = 13) or i.m. (n = 15). Mean [IQR (25; 75)] serum hCG levels in the s.c. and i.m. groups were 171.7 [27.0; 207.0] and 142.2 [102.5; 157.5] mIU/mL, respectively. No adverse events were registered by any patient receiving hCG by either injection method. In this non-IVF population, two pregnancies were established in each subgroup (4 of 28, or approximately 14% pregnancy rate).The s.c. administration of 5,000 IU hCG (reconstituted in vol. = 0.5 mL) was well tolerated by all women in this study and was associated with postinjection serum hCG levels similar to those observed after administration of an equivalent i.m. hCG dose. This investigation suggests that clinical use of s.c. hCG is suitable for lean women (e.g., BMI30) undergoing ovulation induction, but additional data are needed to study the appropriateness of s.c. hCG administration in heavier patients.