Patrick Quinn

Enhanced results in mouse and human embryo culture using a modified human tubal fluid medium lacking glucose and phosphate

PurposeBecause various recent studies in both human and laboratory animals have indicated an influential role of glucose and phosphate in embryonic development, this study was initiated to assess the effect of culture medium lacking glucose and phosphate on human IVF.ResultsInitial studies with mouse zygotes indicated a significant improvement in embryonic development in both a F1 hybrid and a random-bred “two-cell block” strain, using a modified HTF medium which contained EDTA and glutamine (Gln) and also with a similar medium which lacked glucose and phosphate. Subsequently, a prospective randomized trial of human IVF has begun in which infertile patients are sequentially assigned to have IVF using one of three media. The three media are regular HTF, HTF containing EDTA and Gln (referred to as Basal HTF), and HTF with EDTA and Gln but devoid of glucose and phosphate ions (referred to as Basal XI HTF).ConclusionsInitial results indicate that Basal XI HTF medium, a modified form of HTF containing EDTA and glutamine but devoid of glucose and phosphate, greatly improves the in vitro development of mouse and human embryos over HTF and Basal HTF medium.

Successful human in vitro fertilization using a modified human tubal fluid medium lacking glucose and phosphate ions

OBJECTIVE To determine the effect of medium with or without glucose and phosphate on the fertilization and development of human oocytes. DESIGN Sequential allocation of alternate patients to one of two treatment groups. SETTING Private practice infertility programs. PATIENTS Ten couples requesting treatment for infertility. INTERVENTIONS Gametes from each couple were collected, washed, and incubated in one of two culture media under investigation. MAIN OUTCOME MEASURES Number of oocytes collected, fertilized, cleaving, replaced, and implanting in each patient. Development of any supernumerary embryos to fully expanded blastocysts in vitro. RESULTS There was a significant increase in the proportion of transferred embryos implanting in the group of patients whose gametes were handled in medium devoid of glucose and phosphate. All other comparisons of factors that may have influenced implantation rates between the two groups of patients were not significantly different. CONCLUSIONS High rates of fertilization, cleavage, implantation, and development of supernumerary human embryos to the blastocyst stage in vitro were obtained with a modified human tubal fluid medium containing ethylenediaminetetraacetic acid and glutamine but devoid of glucose and phosphate ions. A prospective randomized trial is necessary to evaluate the clinical significance of these observations.

Beneficial effects of coculture with cumulus cells on blastocyst formation in a prospective trial with supernumerary human embryos.

AbstractPurpose: We reported previously on the use of coculture with cumulus cells in insemination medium for the development of human embryos in vitro. Here we describe a prospective trial to determine if this procedure has a significant beneficial effect. Methods: On the day after insemination, zygotes were randomized for culture in either a fresh drop of medium without (− cum) or were left in their insemination drop with (+ cum) cumulus cells. Embryos with the best morphological quality were replaced on the third day of development at the eight-cell stage. The remaining embryos were cultured for a further 3 days and cryopreserved if they reached the fully expanded blastocyst (FEB) stage. Three different culture media were used over the period of this study. Results: In 11 patients, supernumerary embryos were available only for continued culture in + cum and three patients had embryos cultured in only − cum. Thirty-nine other patients had embryos assigned to both + cum and − cum treatments. In the + cum group, 98 blastocysts developed from 216 embryos cultured for 6 days (45%), and this was significantly greater (P<0.01) than the 48 blastocysts from 156 embryos (31%) developing in the absence of cumulus cells. In basal HTF medium (HTF medium with EDTA and glutamine) and basal XI HTF medium (similar to basal HTF but devoid of glucose and phosphate), culture of embryos with cumulus cells produced significantly more FEBs than in the absence of cumulus cells. There was no significant difference between the two culture treatments when regular HTF medium was used. Preliminary results indicate that pronectin-coated dishes provide a good substratum for cumulus cell attachment and embryo development. Conclusions: The culture of human embryos with their cumulus cells in insemination drops of medium produces a significantly greater proportion of FEBs than when the zygotes are transferred to fresh culture drops devoid of cumulus cells. This is the first report of a significantly higher blastocyst rate with coculture in which a real comparison has been made between two culture treatments which differ only in the presence or absence of homologous cumulus cells in insemination drops.

Confirmation of the beneficial effects of brief coincubation of gametes in human in vitro fertilization

OBJECTIVE To confirm whether brief exposure of human oocytes to spermatozoa in vitro results in equivalent fertilization rates and possibly better quality embryos than overnight coincubation and to determine if there was a difference in outcome with regard to the type of culture medium used. DESIGN Prospective distribution of gametes between treatments in sequential patients. SETTING Assisted reproductive technology program in private hospital. PATIENT(S) Consecutively treated subfertile couples entering an infertility program. INTERVENTION(S) Assisted reproductive technology treatment for infertility involving oocyte retrieval and in vitro fertilization. MAIN OUTCOME MEASURE(S) When possible, the outcome of fertilization and embryo quality were compared when gametes were coincubated for 1 hour or overnight. Two different formulations of human tubal fluid were compared in some cases. RESULT(S) There was no statistically significant difference in fertilization rates between a brief or overnight coincubation of gametes or between the two treatment groups with regard to the type of culture medium used. The quality of the embryos was significantly better in the 1-hour exposure group. The embryos in Basal XI human tubal fluid medium were of significantly better morphological quality than their siblings in D3+ human tubal fluid medium. CONCLUSION(S) Coincubation of oocytes and spermatozoa for a shorter period produced embryos of superior morphological quality than the generally accepted overnight protocol. A simple glucose and phosphate-free human tubal fluid medium resulted in early cleavage embryos of better morphological quality than a medium supplemented with glucose, taurine, and glutathione.

Improvement in early human embryo development using new formulation sequential stage-specific culture media

OBJECTIVE To determine whether altering selected components of sequential culture media can improve early development variables of human embryos. DESIGN Prospective, randomized, sibling oocyte split trial. SETTING Private ART center. PATIENT(S) Two hundred eight undergoing treatment with in vitro fertilization or microinjection. INTERVENTION(S) Oocytes from each patient were randomly allocated to fertilization and cleavage media of a control and a trial culture medium formulation. MAIN OUTCOME MEASURE(S) Rates of fertilization, cleavage, and uncontrolled division; average embryo morphology score; blastomeres per embryo; embryo score parameter (number of blastomeres x embryo morphology grade); and embryo utilization. The trial media resulted in a higher fertilization rate, higher cleavage rate, lower rate of uncontrolled division, higher number of blastomeres per embryo, higher average embryo morphology score, a higher embryo score parameter, and higher embryo utilization rate compared to the control media. All differences were statistically significant. CONCLUSION(S) Improved sequential stage-specific culture media can reduce the occurrence of severe human embryo fragmentation and improve developmental variables in early IVF- and ICSI-generated embryos.

Use of coculture with cumulus cells in insemination medium in human in vitro fertilization (IVF)

PurposeIn an initial trial, 16 of 33 (48%) bipronuclear human zygotes left in culture in the insemination drop from which they had originated developed to fully expanded blastocysts.ResultsThis method was subsequently used for all supernumerary embryos judged unsuitable for replacement or cryopreservation on Day 1, 2, or 3 of development. Over a 4-year period, embryos reaching the fully expanded blastocyst stage were cryopreserved. Of 113 such blastocysts thawed, 81 survived (72%), and upon transfer to 52 patients, 8 clinical pregnancies were established (15%), of which 6 were live births. Subsequently, following modification of some culture parameters, 60 patients had 296 supernumerary embryos cultured for 6 days; 43 of these patients (72%) had 148 embryos (50%) that cavitated and 134 (45%) of these cavitating embryos were judged to be fully expanded blastocycts; 125 (42%) of these embryos were cryopreserved.ConclusionThe blastocyst formation rate is similar to that reported by others using conventional culture procedures or coculture on Vero or other cell types. I conclude that cumulus cells are a ready source of feeder cells for the coculture of human embryos.

Suboptimal laboratory conditions can affect pregnancy outcome after embryo transfer on day 1 or 2 after insemination in vitro

We have demonstrated that the replacement of pronuclear stage human embryos on the day after insemination in IVF-ET results in a pregnancy rate as good as, if not better than, the rate after ET on day 2. The pregnancy rate appears to be influenced by the culture environment used for the gametes. We would recommend that ET be undertaken as soon as possible after insemination on day 1 so that if suboptimal culture conditions are present, they do not have sufficient time to affect the viability of the embryos and reduce the subsequent chance of pregnancy after replacement. If culture conditions have been optimized there is no difference in pregnancy rate after embryo transfer on day 1 or day 2 and one can then replace embryos on either day and expect the pregnancy rate obtained in either case to be similar.

Responses of patients to different lots of human menopausal gonadotropins during controlled ovarian hyperstimulation

Responses of patients treated with different lots of human menopausal gonadotropin (hMG) during controlled ovarian hyperstimulation were analyzed. Levels of luteinizing hormone (LH) in serum varied between groups of patients treated with different hMG lots, serum follicle-stimulating hormone (FSH) levels did not differ. In the analysis of levels of estradiol (E2) in serum of patients pretreated with leuprolide acetate (gonadotropin-releasing hormone analog; GnRH-a), there was an interaction between hMG lot and day of stimulation. E2 levels/follicle also diverged between hMG batches as ovum pick-up approached. Within the groups of patients pretreated with GnRH-a, serum FSH/LH ratios varied between 5 and 20, with a batch x day interaction. Ongoing pregnancy rates in the hMG-treatment groups ranged between 0/24 and 7/33 (21%).

Levels of lnterleukin-2 Receptor in Serum of Patients During the Menstrual Cycle and Following Embryo/Gamete Transfer

ABSTRACT: In an attempt to establish whether nidation elicits a measureable systemic immunologic response in vivo, levels of interleukin‐2 receptor (IL‐2R) have been determined in serum drawn from 34 pregnant patients (IUP) immediately before embryo/ gamete transfer, and 7 and 14 days later. For comparison, these same values were determined for 41 subjects who did not conceive, and for 18 who spontaneously aborted (SAB) during the first trimester of gestation. Serum IL‐2R values were near 320 U/ml and did not differ between days within outcomes, or within days between outcomes. Furthermore, levels of IL‐2R and β‐hCG in serum were not interrelated on days 7 or 14 of pregnancy (SAB or IUP). Serum IL‐2R levels did not change during the menstrual cycle. The present results appear to traverse presumptive elements of existing hypotheses of “placental immunotropism.”

Gonadotropin and Estradiol Levels During Ovarian Stimulation in Women Treated With Leuprolide Acetate

&NA; Levels of FSH, LH, and estradiol (E2) were measured in the serum of 209 gonadotropin‐releasing hormone analogue‐treated women and in 202 control subjects during the final 5 days of ovarian stimulation in our in vitro fertilization program. Levels of FSH and E2 in serum of gonadotropin‐releasing hormone analogue‐treated subjects significantly exceeded control values during the sampling period, whereas LH levels were significantly lower. Concentrations of E2 in serum of gonadotropin‐releasing hormone analogue‐treated and control subjects were similar when corrected for differences in numbers of follicles aspirated at oocyte retrieval (mean of 8.9 and 7.2 follicles per subject, respectively). Pregnancy rates by diagnostic ultrasound were 18 and 11%, respectively, a statistically significant difference. (Obstet Gynecol 73:990, 1989)