Human In Vitro Fertilization Research Articles

ORIGINAL ARTICLE: Th1 and Th2 in Human IVF Pregnancy with Allogenic Fetus

Problem Positive obstetric outcome of allogenic in vitro fertilization (IVF) pregnancies makes interesting the subject of additional regulatory mechanisms after oocyte donation.Method of study Eighty eight women: 23 donation of oocytes (DO), 33 IVF, 32 natural conception (NC) were studied in first trimester of pregnancy. Intracellular production of cytokines and chemokine receptors expression were studied by flow cytometry.Results Intracellular production of interferon‐γ (IFN‐γ), interleukin (IL)‐4, tumor necrosis factor‐alpha by CD4 T lymphocytes in DO women was higher than in IVF and NC women. Ratio IFN‐γ/IL‐4 in DO was lower than in IVF. We found higher expression of chemokine receptor CCR4 but not CXCR3 on CD4 T cells in DO compared with IVF and NC. Ratio CXCR3/CCR4 in DO was lower than in NC.Conclusion Hyperactivation of T helper 1 (Th1) and T helper 2 (Th2) by allogenic fetus is specific for DO pregnancy. Preferable activation of Th2 and relative suppression of Th1 chemokine expression reflect additional regulatory counteractive mechanism(s).

Effects of Extracellular Adenosine 5′-Triphosphate on Human Sperm Motility

Extracellular adenosine 5'-triphosphate (ATP) previously has been shown to increase the fertilization percentage in human in vitro fertilization (IVF) performed for male factor infertility. The objective of this study is to determine the effects of extracellular adenosine 5'-triphosphate (ATPe) on human sperm function by examining its effects on end points of sperm capacitation. Sperm obtained from healthy volunteers with normal semen parameters, asthenozoospermic men, and cryopreserved samples were incubated in medium with or without 2.5 mM ATPe. The effects of ATPe on acrosomal exocytosis, protein tyrosine phosphorylation, and sperm motility parameters were quantified. Although ATPe did not affect acrosomal exocytosis or protein tyrosine phosphorylation in sperm from healthy donors, it significantly altered several motility parameters, with the largest effects manifested in increased curvilinear velocity and percentage hyperactivation. ATPe similarly affected sperm selected for poor motility and thawed cryopreserved sperm but to a lesser extent than its effects on sperm with normal motility. ATPe increased straight-line velocity and linearity of sperm obtained from asthenozoospermic men. Human sperm motility characteristics are altered by ATPe; this finding may explain its previously reported beneficial effect on human IVF. These results suggest that ATPe could constitute a new therapeutic modality in the treatment of male infertility.

Sperm protamine 1/protamine 2 ratios are related to in vitro fertilization pregnancy rates and predictive of fertilization ability

To evaluate whether aberrant sperm P1/P2 ratios are predictive of abnormal fertilizing ability and are related to in vitro fertilization (IVF) outcome. Prospective case-control study. University-based infertility and IVF clinic. Forty-three male infertility patients with an abnormally reduced P1/P2 ratio, 251 patients with a normal P1/P2 ratio, and 121 patients with an abnormally elevated P1/P2 ratio. Human IVF, the sperm penetration assay (SPA), and sperm protamine quantification via nuclear protein extraction, gel electrophoresis, and densitometry analysis. Sperm P1/P2 ratios; P1 and P2 quantities; SPA scores; and IVF-fertilization, embryo-quality, pregnancy, delivery, and spontaneous-abortion rates. Standard IVF fertilization rates and SPA scores were significantly reduced in patients with abnormally low and high P1/P2 ratios. In vitro fertilization embryo quality was comparable between these groups, but pregnancy rates were significantly reduced in patients with abnormally reduced P1/P2 ratios. The P1/P2 ratio has a significant relationship to sperm fertilization ability. The relationship between protamines and fertilization ability is not understood but may be either a reflection of generalized abnormalities during spermiogenesis or an indication of protamine deficiency acting as a regulator or checkpoint of spermatogenesis.

Comparison of Blastocyst Formation Between Standard and Clomiphene Citrate (CC) Stimulated In Vitro Fertilization (IVF) Cycles

Many studies have shown that single blastocyst transfer can achieve pregnancy rates comparable to that of multiple embryo transfer. Nonetheless controlled ovarian hyperstimulation (COS) with daily injection of gonadotropins designed to produce large numbers of eggs is the most commonly employed protocol in human IVF. The aim of current study is to determine whether a less expensive, minimal stimulation IVF protocol using clomiphene citrate (CC) in combination with a small addition of human menopausal gonadotropin (hMG), yielding only a modest number of eggs, will produce blastocysts with success rates similar to standard protocols which are designed to yield many eggs. Prospective and randomized. Patients enrolling for IVF were randomized into two groups. Both groups received one cycle of oral contraceptives (OCP’s) before commencement of IVF stimulation. Group 1, clomiphene citrate protocol (CC-IVF): daily clomiphene citrate 50 mg was initiated orally on day 3 after OCP’s until gonadotropin releasing hormone agonist (GnRHa) nasal spray (Synarel) was administered. hMG 150 IU subcutaneous(SQ) every 48 hours was begun on day 8. Group 2, microdose GnRHa (Lupron) flare protocol (Lupron-IVF): Lupron 25 mcg SQ was begun on cycle day 3 after OCP’s followed by daily recombinant follicle stimulating hormone (rFSH) 150-225 IU plus human menopausal gonadotropin (hMG) 75-150 IU SQ on day 5 after OCP’s. Final oocyte maturation was triggered by GnRHa nasal spray (Synarel) in the CC-IVF protocol, and by 250 mcg recombinant hCG in the Lupron-IVF protocol. Follicular development was monitored by transvaginal ultrasound as well as plasma estradiol and LH levels. All monitoring and oocyte retrievals were performed by the same physician and all IVF procedures were performed by the same embryology team within the same period of the time. Statistical analysis was performed using t-test and chi square analysis. As displayed in the table dramatically less gonadotropin was used in the CC-IVF cycles than the Lupron-IVF cycles. While the average numbers of oocytes obtained and subsequent number of blastocysts were higher in Lupron-IVF cycles, the blastocyst formation rate was similar between the CC-IVF cycles and the Leupron-IVF cycles. Tabled 1 The minimal stimulation protocol with clomiphene citrate and hMG produces fewer oocytes as compared with a standard protocol using daily injection of rFSH, hMG and GnRHa. However, the oocytes obtained from the minimal stimulation protocol have similar potential to generate blastocysts. Minimal stimulation simplifies IVF procedures, and reduces the incidence of hyperstimulation syndrome. Single blastocyst transfer with minimal ovarian stimulation will also reduce the risk of multiple pregnancy and its complications.

The expression of matrix metalloproteinase‐9 in human follicular fluid is associated with in vitro fertilisation pregnancy

To investigate the role of matrix metalloproteinase-9 (MMP-9) in the pre-ovulatory follicular fluid and culture media during in vitro fertilisation (IVF) cycle and to develop the zymographic pre-diagnosis marker for successful implantation and pregnancy in human IVF. Controlled clinical study. IVF Laboratory, Women's Hospital Infertility Clinic and Dongguk University, Korea. Women undergoing in vitro fertilisation treatment. Experiments were designed for controlled clinical study with women undergoing IVF treatment. MMP-9 expressions in follicular fluid and culture media samples that had been collected during transvaginal oocyte retrieval were measured using zymography. MMP-9 activities and expressions were strongly correlated to a higher rate of fertilisation and pregnancy. Fertilisation rates and ultrasonic evidence of intrauterine pregnancy by four weeks after embryo transfer. MMP-9 activity was significantly higher in the pregnant group than in the non-pregnant group (P < 0.01). In contrast, MMP-2 activity was present in the follicular fluid and culture media of all women, and no difference in its expressions was found between the pregnant and non-pregnant groups. No correlation was found between the MMP-9 expression in follicular fluid and culture media and the fertilisation rates. The expression of MMP-9 in the follicular fluid and culture media is a prerequisite for successful pregnancy in IVF cycle. The zymography of MMP-9 activity in follicular fluids of human and culture media was developed as a pre-diagnostic method and zymographic diagnosis marker for successful fertilisation, implantation and pregnancy in human IVF.

Quality Control in Human In Vitro Fertilization

The implementation of suitable quality control (QC) is not only required for the accreditation of a human in vitro fertilization (IVF) laboratory, but is also fundamental to its success. Several assays have been employed to screen culture media and contact supplies. The suitability of one assay in particular, the mouse embryo assay (MEA), has been questioned over the years. Here we discuss how the conditions of such an assay, together with the stage of embryonic development used, have a profound effect on the outcome of the assay. Furthermore, by assessing embryos at multiple time points during the preimplantation period (rather than simply determining blastocyst formation), together with quantitating key parameters such as blastocyst cell number, it is possible to identify suboptimal components of a culture system. As well as identifying those components that result in outright embryonic demise, under the appropriate conditions the MEA can detect components that lead to impaired development. It is proposed that under the appropriate conditions, the MEA is a useful adjunct to quality control in human IVF, but several assays used in concert are better than a single test.

Effects of culture conditions on IVF outcome

Although in vitro fertilization (IVF) success rates have improved over the past decade, multiple pregnancies have become a formidable problem. The solution to this problem seems simple by mandating the reduction in numbers of embryos transferred. However, this is typically not accomplished without a compromise in the pregnancy rate. There have been a number of approaches designed to address high order multiple pregnancies from multi factorial analysis of early cleavage stage embryos to the development of extended culture systems, both of which require manipulations in the culture environment. Manipulations in embryo culture environment may not be benign. Several studies have demonstrated that adverse culture conditions have effects on gene expression and imprinting. Studies have also demonstrated that singleton human IVF babies have lower birth weight and higher incidence of congenital anomalies than natural conception babies. All of these factors need to be considered in relation to long term viability of IVF babies and the Barker hypothesis.

Characterization of semen from olive baboons.

Electroejaculation is an accepted method of semen collection from non-human primates and is typically performed using direct penile or rectal probe electro-stimulation. Six mature male olive baboons (Papio anubis) were subjected to rectal probe electro-stimulation procedure that yielded 65 usable ejaculates in 69 attempts over a period of 16 weeks. The four non-usable ejaculates consisted of seminal fluid without sperm cells and thus were not analysed. The analysis of ejaculate volume, concentration, % motility, liquefaction times, and morphology in this study has provided information on the quality of semen samples. It is anticipated that this analysis will provide valuable information for male fertility studies and the development of an in vitro fertilization (IVF) system in the olive baboon as a model for human IVF.

Analysis of factors associated with multinucleate formation in human IVF.

This study was carried out to analyse if some of the sperm parameters assessed by computer-aided sperm analysis (CASA) are associated with multinucleate formation in human in vitro fertilization (IVF). In the present study, 137 IVF-embryo transfer (ET) treatment cycles in which pronucleate formation could be observed were enrolled. Sperm parameters after swim-up were assessed using CASA and strict criteria. Thirty-two (2.3%) of 1368 oocytes fertilized were judged as multinucleate formation, which occurred in 25 of 137 IVF-ET treatment cycles, giving the incidence of 18.2%. The factors associated with multinucleate formation were analysed. There were significant differences of the numbers of oocytes collected (13.9 vs. 9.2; p=0.028), fertilization rate (88.8% vs. 71.9%; p=0.011), curvilinear velocity (VCL) (162.7 microm/sec vs. 149.1 microm/sec; p=0.034), average path velocity (VAP) (100.3 microm/sec vs. 93.1 microm/sec; p=0.016), and amplitude of lateral head displacement (ALH) (5.9 microm vs. 5.3 microm; p=0.046) between 25 cycles with multipronucleate formation and 112 cycles with two pronucleate formations. However, there were no significant differences of women's age or the number of sperm inseminated between the two groups. These findings suggest that prediction of multinucleate formation in human IVF might be possible in patients when the number of oocytes collected is higher and sperm motility parameters after swim-up are faster. However, prevention of multinucleate formation seems to be unnecessary because a higher successful IVF-ET outcome is expected as a result of the excellent fertilization rate in these patients.

Using the male gamete for assisted reproduction: past, present, and future.

Using the male gamete for assisted reproduction: past, present, and future.

Blastocyst culture and transfer: a step toward improved in vitro fertilization outcome

Objective: To evaluate the efficacy of blastocyst culture and transfer in human in vitro fertilization (IVF) as compared to day 3 embryo transfer. Design: Prospective randomized trial. Setting: Private assisted reproduction unit. Patient(s): A total of 162 IVF patients were included in the day 3 embryo transfer (n = 82) and blastocyst transfer (n = 80) groups. Intervention(s): Embryo transfer on day 3 after culture in the standard culture media and blastocyst transfer on day 5 or 6 after culture in the sequential culture media. Main Outcome Measure(s):Implantation and pregnancy rates, multiple gestation rate. Result(s): The implantation rate for embryos transferred at the blastocyst stage was significantly higher than that for embryos transferred on day 3 (26% vs. 13%). The viable pregnancy rate was similar in both groups (29% vs. 26%). Significantly fewer embryos were required for transfer at the blastocyst stage compared with day 3 embryo transfer (2.0 ± 0.1 vs. 3.5 ± 0.63). The high-order multiple gestation rate was significantly less with the blastocyst transfer than with the day 3 embryo transfer (4% vs. 19%). Conclusion(s): With the use of blastocyst culture, a few embryos can be transferred without decreasing the overall pregnancy rate. This may reduce multiple gestations and improve human IVF outcome.

Simple improvements of culture systems and selection criteria of viable blastocysts (B) could increase success rates in clinical in vitro fertilization (IVF).

Objective: Human IVF with B transfers has been proved to increase implantation rates but optimal preimplantation embryo (PE) culture conditions and objective criteria for B quality noninvasive evaluation are currently lacking (Fertil Steril 74:828:29). The objectives of this study were: (1) to determine the ability of different culture systems to support poor quality PE development to trophectoderm outgrowth stage (TOS), (2) to determine which objective parameters of B quality could predict its viability (known to correlate positively with the number of nuclei per B, for instance) prior to the uterine transfer.

Reproduction in the Noughties: will the scientists have all the fun?

The past 20–30 years have seen major advances in our understanding of human reproduction and in our ability to manipulate it, as well as major social changes in human reproductive and sexual attitudes. Many of these advances and changes developed out of the first successful in vitro fertilisation (IVF) of the human oocyte. It is also the case that 30 years ago few foresaw what was to come, and many were at best doubtful and often were very critical of the scientific work which led to human IVF and to many of the subsequent developments. This lack of foresight provides us with a lesson about the dangers that we face in looking forward and attempting to predict the future. This review will try to convey, not comprehensively but through examples, the flavour of current activities in Assisted Reproduction clinics and research laboratories around the world and what is being talked about for the future in respect of emergent patient demands and anticipated clinical needs. This clinically driven approach will form the basis for consideration of some underlying scientific aspects of reproductive research, some of the ethicolegal issues that may arise, and the implications of this anticipated future for our current approaches to medical education. The future will be considered not simply in terms of the New Reproduction itself but also in its interaction with the opportunities and challenges presented by the New Genetics. It is perhaps in the interaction between these two fields of endeavour that some of the most difficult challenges ahead lie.

Reproduction in the Noughties: will the scientists have all the fun?

The past 20-30 years have seen major advances in our understanding of human reproduction and in our ability to manipulate it, as well as major social changes in human reproductive and sexual attitudes. Many of these advances and changes developed out of the first successful in vitro fertilisation (IVF) of the human oocyte. It is also the case that 30 years ago few foresaw what was to come, and many were at best doubtful and often were very critical of the scientific work which led to human IVF and to many of the subsequent developments. This lack of foresight provides us with a lesson about the dangers that we face in looking forward and attempting to predict the future. This review will try to convey, not comprehensively but through examples, the flavour of current activities in Assisted Reproduction clinics and research laboratories around the world and what is being talked about for the future in respect of emergent patient demands and anticipated clinical needs. This clinically driven approach will form the basis for consideration of some underlying scientific aspects of reproductive research, some of the ethicolegal issues that may arise, and the implications of this anticipated future for our current approaches to medical education. The future will be considered not simply in terms of the New Reproduction itself but also in its interaction with the opportunities and challenges presented by the New Genetics. It is perhaps in the interaction between these two fields of endeavour that some of the most difficult challenges ahead lie.

Growth of human preimplantation embryos in vitro

The human oocyte fertilizes and develops into embryos in the Fallopian tube and reaches the uterus only after compaction. However, for several years embryos that were developed following in-vitro fertilization (IVF) were transferred into the uterus on day 2 or 3 at the 4–8 cell stage in contrast to the in-vivo situation where they would be present in the Fallopian tube. Earlier attempts to grow embryos in vitro for 5 to 6 days were not always successful. Attempts were therefore made to understand the in-vivo environment of the Fallopian tube where the early embryonic development occurs. This article reviews the studies carried out to understand the composition of fluids in the Fallopian tube specifically with reference to the energy metabolites – lactate, pyruvate and glucose; it also covers how the formulation of culture media for human IVF and embryonic development were modified over the years based on some classical work done on embryo culture in laboratory animals.

Early human development.

This presentation covers original research conducted since 1977 on excess human embryos obtained from several invitro fertilization (IVF) centres around the world. Most of the embryos portrayed were developed from normal, monospermic, bipronuclear ova or from dispermic, tripronuclear ova, after fertilization. With respect to morphology, early dispermic embryos resemble normally fertilized embryos, although they are genetically triploid. The process of human fertilization has been already presented on CD-ROM. Two visual atlases have been published recently on human IVF and embryo development (Sathananthan et al., 1991; Sathananthan, 1996). The preimplantation development of the human embryo begins at fertilization and ends at the implantation of the blastocyst in the uterine endometrium on day 6 after fertilization. Preimplantation development occurs in the Fallopian tube or oviduct and in the uterus during which time the embryo cleaves by repeated mitosis to form a hollow blastocyst. There is little growth and differentiation during cleavage and the embryo is partly dependent on the secretions produced by the oviduct and uterus, since the human egg has no yolk. The embryonic genome is thought to be activated between the 4–8-cell stages of development and the first signs of cell differentiation are evident in the blastocyst, when an outer trophoblast and an inner cell mass or embryoblast have formed. Recent work suggests that somatic and germline cells may be allocated from the 4-cell stage (Edwards and Beard, 1997). The blastocyst finally hatches out from the zona pellucida, or shell, within the uterus and immediately invades the uterine endometrium by means of its trophoblast, after which it is dependent on the mother’s blood for sustenance and eventually develops a placenta. Molecular events during human implantation, especially those involving embryonic interleukins and epithelial β3 integrins, are described by Simon et al. (1998). Human embryos can be developed to blastocysts and even to hatching in vitro. This process of hatching is one of the wondrous events of early development. Embryos are normally developed to the 2–4-cell stages for embryo transfer during IVF and are replaced either in the uterus or Fallopian tube.

Development of serum-free media for the culture and transfer of human blastocysts.

The culture and transfer of the blastocyst stage embryo has several advantages for assisted reproduction in the human. However, due to inadequacies of present culture conditions in human in-vitro fertilization (IVF), embryos are routinely transferred to the uterus on either day 2 or day 3 of development around the 4- to 8-cell stage, with resultant implantation rates of only 10-25%. In other mammalian species the transfer of cleavage stage embryos, which normally reside in the oviduct, results in a significantly lower implantation rate compared with the transfer of blastocysts. Extended culture of human embryos in vitro will help to identify those embryos with little, if any, developmental potential. It is therefore plausible that the blastocyst has an intrinsically higher viability than the cleavage stage embryo. It has now been shown in human IVF that sequential serum-free media can support > 50% blastocyst development, with an implantation rate per blastocysts of 50%, double that obtained for cleavage stage embryos. As the implantation rate of the blastocyst is higher than the cleavage stage embryo, fewer blastocysts are required for transfer. The development of completely defined embryo culture media may prove feasible by the replacement of protein with the glycosaminoglycan hyaluronate. Hyaluronate, which is protein-free, is more suitable than albumin in supporting implantation in the mouse, and can eliminate the biological variation inherent when using protein and the potential for contamination when using blood products such as albumin.

Optimization of culture conditions for human in vitro fertilization and embryo transfer.

Approximately 20 years ago the first child conceived with in vitro fertilization (IVF) and embryo transfer was born in England. Although overall pregnancy rates and delivery rates after IVF have improved over the years, success between IVF clinics can vary as much as tenfold. The factors that influence the success rate include type of patient, ovarian stimulation protocol, quality of oocytes, culture conditions, handling of gametes and embryos, quality of embryos, embryo transfer technique, and endometrial receptivity. Culture conditions and handling of gametes and embryos will be reviewed with the hope of improving success of human IVF. The ongoing clinical pregnancy rates in our program increased from about 30% in 1995 and 1996 to about 50% in 1997. Improved embryo culture conditions and embryo cryopreservation technology should not only increase fresh but also frozen-thawed embryo transfer pregnancy. This will make IVF efficient and cost-effective while achieving high overall pregnancy rates with lower multiple pregnancy rates.

Culture of viable human blastocysts in defined sequential serum-free media.

In human in-vitro fertilization (IVF), embryos are routinely transferred to the uterus on either day 2 or day 3 of development, resulting in a 10-15% implantation rate. However, in other mammalian species, the transfer of cleavage stage embryos, which normally reside in the oviduct, to the uterus results in a significantly lower implantation rate compared with blastocysts. It is therefore proposed that, in order to increase implantation rates in human IVF, one has to move to extended culture and transfer at the blastocyst stage. The transfer of blastocysts will not only help synchronize the embryo with the female tract but will facilitate the identification of those embryos with little or no developmental potential. In order to culture viable blastocysts it is important to use more than one culture medium to cater for the changing requirements of the preimplantation embryo as it develops and differentiates. If sequential culture media are not used, one can obtain blastocysts but their resultant viability is low. The use of sequential serum-free media in human IVF has resulted in > 50% of embryos becoming blastocysts with an implantation rate of approximately 50%. Further advances in human embryo culture should come from the replacement of protein with the glycosaminoglycan hyaluronate, which is more suitable than albumin in supporting implantation in the mouse, and which will eliminate biological variation and possible contamination from blood products. With the routine culture of human blastocysts will come the introduction of non-invasive tests of embryo viability, capable of identifying those blastocysts most likely to develop from a given cohort. As the implantation rate of blastocysts is higher than that of the cleavage stage embryo, fewer embryos will be required for transfer in order to establish a successful pregnancy, thereby reducing the number of multiple gestations and increasing the overall efficiency of human IVF.

Effect of maternal age and conditions of fertilization on programmed cell death during murine preimplantation embryo development.

One of the major morphological anomalies observed in many human pre-embryos is extensive cellular fragmentation. Previously we confirmed that embryo fragmentation seemed to be associated with the activation of programmed cell death (PCD). The purpose of our experiments was to establish a rate for murine embryo fragmentation in vivo after hormonal stimulation in young versus older females and to compare it with the rate of embryo fragmentation during in-vitro fertilization (IVF). While murine maternal age beyond 40 weeks increased the rate of embryo fragmentation following in-vivo fertilization (P = 0.001), oocytes from females of all ages had a uniformly high rate of fragmentation when fertilized in vitro (33%). None of the fragmented murine embryos proceeded further in development. In the mouse, fragmentation occurs exclusively during the first cell cycle. Furthermore, IVF significantly reduced the rate of blastocyst formation (P = 0.0001) and decreased the mean cell number at the blastocyst stage in comparison with embryos produced in vivo (P < 0.0001). The cell death index was significantly affected by both maternal age (P = 0.005) and IVF (P = 0.0001). Identification of specific factors which trigger PCD, especially those associated with IVF, may enable us to lower the rates of fragmentation in preimplantation embryos and thereby increase pregnancy rates after human IVF.