In Vitro Fertilization Trials Research Articles

Exogenous progesterone during in vitro fertilization improves developmental competence of partially cumulus-denuded bovine oocytes

The progesterone (P4) secreted by cumulus cells has gained attention for its role as a possible physiological inducer of sperm acrosome exocytosis. In mammals, it is generally accepted that fertilization rates of oocytes without cumulus are markedly low. This study assessed the integrity of capacitated bovine sperm acrosome when exposed to increasing concentrations of P4, and evaluated whether exogenous P4 during in vitro fertilization (IVF) increases the developmental competence of partially cumulus-denuded oocytes in serum-free conditions. After a 4-h capacitation induction, sperm were incubated with increasing concentrations of P4 (0, 0.1, 10 and 100 μM) and evaluated for viability, caspase activation and acrosome status at three different times (4, 5, and 22 h), including the capacitation induction period. Progesterone induced sperm acrosomal exocytosis without compromising sperm viability or activating sperm caspases. Sperm undergoing acrosome reaction exhibited three differential Concanavalin A patterns, corresponding to early, intermediate and late acrosomal exocytosis. The percentage of these patterns significantly increased over time, regardless of P4 concentration, except for those spermatozoa with late acrosomal exocytosis, which only showed an increase at 22 h of incubation. After incubation for 1 h with 100 μM P4, spermatozoa showing intermediate acrosomal exocytosis significantly increased. At 22 h of incubation, the pattern corresponding to early acrosomal exocytosis evidenced a dose-dependent increase. However, prematurely high levels of acrosome reaction induced by 100 μM P4 led to inefficient IVF outcomes (P < 0.05). Therefore, IVF trials with partially cumulus-denuded oocytes were carried out with lower P4 concentrations (0, 0.1, 1, 5, 10 μM). Cleavage rate significantly increased at 1 μM P4, which translated to increased total embryo production after 7 days of in vitro culture (P < 0.05). Significantly higher percentages of expanded blastocysts were observed at both 1 μM and 10 μM P4 as compared to the other experimental conditions. In conclusion, the different patterns of acrosomal exocytosis identified over time by incubation of live sperm with a fluorescent lectin revealed the existence of sperm subpopulations heterogeneous in their physiological states. Moreover, exogenous P4 at 1 μM during IVF improved the developmental competence of partially cumulus-denuded oocytes in serum-free conditions.

Comparison of complication rates after transvaginal ultrasound-guided oocyte pick-up procedures with respect to ovarian response.

ObjectiveThe aim of this study was to compare the complication rates of oocyte pick-up (OPU) procedures via transvaginal ultrasonography in patients with different levels of ovarian reserve. MethodsIn total, 789 patients who underwent OPU procedures for in vitro fertilization (IVF) were included in the study.ResultsIndividuals with normal ovarian reserve had a 2.947-fold higher risk of complications in OPU procedures than individuals with low ovarian reserve, and individuals with high ovarian reserve had a 7.448-fold higher risk of complications than individuals with low ovarian reserve. In addition, a higher number of IVF trials was associated with an increased risk of complications.ConclusionThe results of this study show that OPU has a higher risk of complications, particularly severe pain, in patients with high ovarian reserve. It is thought that complications can be reduced by preferring mild stimulation in patients with high ovarian reserve. Collecting fewer oocytes is also associated with a lower risk of complications from OPU. Even if a patient’s reserve is very good, fewer and higher-quality oocytes should be targeted with the use of the lowest possible dose of drugs.

PSXI-10 How to use ultrasonic cutter on frozen semen for multiple embryo production

Abstract The objective of the present study was to establish conditions for using technology that can potentially enhance the efficiency of bovine embryos derived from in vitro fertilization (IVF) with frozen semen. Frozen semen from selected bulls can be stored indefinitely in liquid nitrogen as genetic resources; however, these resources are considered consumable because they cannot be regenerated. Therefore, to optimize the utilization of frozen semen, as many oocytes as possible should be fertilized with one straw. However, a sufficient number of prepared oocytes might not be available for one experiment, which can limit the use of the total spermatozoa population. Thus, an economical method for producing embryos needs to be established by optimizing technology for transplantable embryos. In this study, the utilization of frozen semen was increased by dividing the straw with an ultrasonic cutter. The post-thaw survival rate of uncut straws from Korean Proven Bulls did not differ from that of half cuttings. When ultrasonic cutting was applied to frozen semen, spermatozoa could be prepared for IVF trials at least four times, and blastocysts were produced. Therefore, cutting frozen semen with an ultrasonic cutter represents a potentially useful tool to expand genetic resources from excellent breeding stocks. This approach could also be valuable in the field of IVF of endangered species or rare breeds for their preservation, as well as in ovum pick-up (OPU) techniques.

Ultrasonic Cutting of Frozen Semen Straws to Optimize the Use of Spermatozoa for In Vitro Fertilization.

Simple SummaryThe use of frozen semen is essential for the success of in vitro fertilization in the bovine embryo transfer industry. Therefore, we aimed to maximize the use of a single frozen semen straw by employing a cutting protocol. To the best of our knowledge, the current study is the first to apply ultrasonic cutting to frozen semen. The mechanical damage of spermatozoa was studied in frozen bovine semen by assaying sperm motility, acrosome abnormalities, and developmental competence. These findings may help to optimize the utilization of frozen semen for in vitro fertilization in the bovine industry.The objective of the present study was to establish conditions for using technology that can potentially enhance the efficiency of bovine embryos derived from in vitro fertilization (IVF) with frozen semen. Frozen semen from selected bulls can be stored indefinitely in liquid nitrogen as genetic resources; however, these resources are considered consumable because they cannot be regenerated. Therefore, to optimize the utilization of frozen semen, as many oocytes as possible should be fertilized with one straw. However, a sufficient number of prepared oocytes might not be available for one experiment, which can limit the use of the total spermatozoa population. Thus, an economical method for producing embryos needs to be established by optimizing technology for transplantable embryos. In this study, the utilization of frozen semen was increased by dividing the straw with an ultrasonic cutter. The post-thaw survival rate of uncut straws from Korean Proven Bulls did not differ from that of half cuttings. When ultrasonic cutting was applied to frozen semen, spermatozoa could be prepared for IVF trials at least four times, and blastocysts were produced. Therefore, cutting frozen semen with an ultrasonic cutter represents a potentially useful tool to expand genetic resources from excellent breeding stocks. This approach could also be valuable in the field of IVF of endangered species or rare breeds for their preservation, as well as in ovum pick-up (OPU) techniques.

Improvement of in vitro fertilization by a tannin rich vegetal extract addition to frozen thawed boar sperm

Boar spermatozoa are very susceptible to cryopreservation injuries and, for this reason, pig remains one of the few species in which fresh semen is still preferred to thawed one for routine artificial insemination (AI). The present work evaluated the effect of supplementing boar sperm thawing medium with Silvafeed SP (SSP), a mixture of Chestnut and Quebracho wood extracts (60/40 w/w) rich in polyphenols (92.4% tannin content) on in vitro fertilization (IVF) and on the following sperm parameters: sperm motility (assessed by CASA), viability, acrosome integrity, mitochondrial function and lipid peroxidation (assessed by flow cytometry) and capacitation status (immunolocalization of tyrosine phosphorylated proteins). Thawed spermatozoa were incubated 1 h at 37°C in BTS without (CTR) or with (5, 10, 20 µg/mL) SSP. After incubation sperm suspension was divided in three aliquots: one was used for IVF trials, one for sperm analysis, and the last one was capacitated for 1 h at 39°C 5% CO2 in IVF medium. Sperm motility parameters, viability, acrosome integrity, mitochondrial functionality, lipid peroxidation and tyrosine phosphorylated protein immunolocalization, used as capacitation parameter, were not influenced by SSP. However, oocytes inseminated with thawed spermatozoa pretreated with all the different SSP concentrations presented a significant (P < 0.01) increase in penetration rate compared to CTR. In addition, 5 µg/mL SSP exerted a positive effect (P<0.05) on the total efficiency of fertilization. These results encourage the use of SSP in the thawing medium since post-thawing fertility is a limit for the large-scale use of boar frozen semen.

Improvement of in vitro fertilization by a tannin rich vegetal extract addition to frozen thawed boar sperm

Boar spermatozoa are very susceptible to cryopreservation injuries and, for this reason, pig remains one of the few species in which fresh semen is still preferred to thawed one for routine artificial insemination (AI). The present work evaluated the effect of supplementing boar sperm thawing medium with Silvafeed SP (SSP), a mixture of Chestnut and Quebracho wood extracts (60/40 w/w) rich in polyphenols (92.4% tannin content) on in vitro fertilization (IVF) and on the following sperm parameters: sperm motility (assessed by CASA), viability, acrosome integrity, mitochondrial function and lipid peroxidation (assessed by flow cytometry) and capacitation status (immunolocalization of tyrosine phosphorylated proteins). Thawed spermatozoa were incubated 1 h at 37°C in BTS without (CTR) or with (5, 10, 20 µg/mL) SSP. After incubation sperm suspension was divided in three aliquots: one was used for IVF trials, one for sperm analysis, and the last one was capacitated for 1 h at 39°C 5% CO2 in IVF medium. Sperm motility parameters, viability, acrosome integrity, mitochondrial functionality, lipid peroxidation and tyrosine phosphorylated protein immunolocalization, used as capacitation parameter, were not influenced by SSP. However, oocytes inseminated with thawed spermatozoa pretreated with all the different SSP concentrations presented a significant (P < 0.01) increase in penetration rate compared to CTR. In addition, 5 µg/mL SSP exerted a positive effect (P<0.05) on the total efficiency of fertilization. These results encourage the use of SSP in the thawing medium since post-thawing fertility is a limit for the large-scale use of boar frozen semen.

Successful in vitro Fertilization Using Autologous Gametes after Eight Failed Trials

Background: Many infertile couples worldwide resort to donated gametes to overcome their infertility problems, however many others cannot for cultural or religious reasons. They have no other options but probably to keep trying In-Vitro Fertilization (IVF) treatment using their own gametes. We wanted to see if we would be able at some point to help such cases fulfil their dreams. Case Report: Four infertility cases with a history of many unsuccessful IVF trials; eight, seven, six, and four, suggestively because of very poor fertilization and/or poor embryo development due to sever male factor that ranged from azoospermia to severe oligo-terato-asthenozoospermia. Yet, in their subsequent trials we managed to help them conceive with live births at the end. Conclusion: In some complex infertility cases, when the use of donor-gametes is not an option, probably persistent hopefulness in IVF treatment with autologous gametes stays as the solution.

Effect of Different Media and Protein Source on Equine Gametes: Potential Impact During In Vitro Fertilization.

Equine in vitro fertilization (IVF) is still inconsistent. In the present work, we studied how modified Whitten's (MW) medium and Tissue Culture Medium 199 (TCM) added with Foetal Bovine Serum (FBS; 10% v/v) or Bovine Serum Albumin (BSA; 7 mg/ml) affected equine gametes to subsequently run IVF trials. Compact (Cp) and expanded (Ex) cumuli equine oocytes were matured and placed in TCM or MW supplemented with BSA or FBS for 18-20 h (no sperm added). In Ex oocytes, TCM-199 added with FBS or BSA resulted in higher metaphase II (MII) rates (75.7% and 62.7%, respectively) than MW added with BSA (54%) or FBS (52.2%; p < 0.05); this was not observed for Cp oocytes. Equine sperm were capacitated in the same media at 10 × 10(6) sperm/ml for 4 h at 37°C; total motility and protein tyrosine phosphorylation (PY) were evaluated. While motility remained unchanged, TCM or MW added with FBS enhanced the number of sperm showing PY-stained tails (25 ± 4.8% and 31 ± 6.6%; mean ± SEM, respectively) over BSA supplemented media (3 ± 1.2% and 11.7 ± 1.1%) for TCM and MW (p < 0.05). In view of the previous results, sperm were capacitated in TCM + FBS and MW + BSA (control); IVF trials were run in the same media supplemented with 200 ng/ml of progesterone, but no fertilization occurred. Our results show that TCM + FBS enhances Ex equine oocyte's meiotic competence over MW + BSA and TCM or MW added with FBS successfully induce equine PY over media supplemented with BSA.

Use of complementary medical therapies by Israeli patients undergoing in vitro fertilization

ObjectiveTo evaluate the prevalence and characteristics of complementary medical therapy (CMT) use among Israeli couples undergoing in vitro fertilization (IVF). MethodsIn a cross-sectional study, men and women undergoing treatment at four IVF units in Israel were invited to complete an anonymous questionnaire between May 2010 and December 2011. Patients were considered users of CMT if they reported that either partner used at least one type of CMT for treating infertility. Stepwise backward logistic regression was used to assess the independent effects of variables on CMT utilization. ResultsOf 511 patients approached, 400 (78.1%) completed the survey and 159 (39.8%) indicated that CMT for infertility was used by one or both partners. Higher CMT use was significantly associated with the treating hospital, post high-school education, more than three previous IVF trials, being employed, and using psychosocial support (all P<0.05). Most users (75/129; 58.1%) did not notify the IVF clinic medical staff about concurrent use of CMT. ConclusionUse of CMTs was widely reported by Israeli patients undergoing IVF, particularly those with higher education, and those undergoing repeated IVF trials and receiving psychosocial support. IVF staff ought to be aware of the widespread utilization of CMTs because the impact of these therapies on IVF outcomes is inconclusive.

Psychoanalytic Understanding of Repeated In-Vitro Fertilization Trials, Failures, and Repetition Compulsion

Rapid advances in reproductive technologies confront us with the need to understand the complex psychological impact on participating parties. Understanding of repetition compulsion and psychoanalytic understanding of repeated IVF trials and failures is an important dynamic in some of the patients who resort to these methods. The discussion of two detailed case histories, illustrate how infertility traumata were re-experienced compulsively during the course of treatment. The unconscious self-induced traumatization resulted from the compulsion to repeat an earlier repressed trauma. The denial of multiple and repeated failures illustrate a complex compilation of several unprocessed losses that the individuals have yet to finish mourning. In addition, the lack of acceptance of a failure to conceive presented itself as a technical challenge offering an understanding of repeated past experiences in the context of the transference and counter transference paradigm. Each new IVF cycle followed the unprocessed mourning of a loss with persistence to repeat. The repeating of the past experiences over and over in a rather fast-paced use of ARTs shows the strong evidence for dynamic repetition compulsion phenomena. The denial of the failure to conceive can linger on, becoming a long process with unfinished mourning throughout the life cycle. There is a need for psychoanalytic thinking and understanding of psychological implications of the use of In-Vitro Fertilization (IVF).

Effects of paternal cigarette smoking on testicular function, sperm fertilizing capacity, embryonic development, and blastocyst capacity for implantation in rats

Summary. We evaluated the effects of paternal smoking on testicular function, sperm fertilizing capacity, embryonic development, and blastocyst capacity for implantation. Rats of group A were exposed to cigarette smoke for 10 weeks. Rats of group B were exposed to the smoke of incense sticks for 10 weeks. Rats of group C served as a control group. Rats of group D were exposed to cigarette smoke for 7 weeks only. Experimental period was 10 weeks in all groups. At the end of the experimental period serum testosterone responses to human chorionic gonadotropin stimulation, andro-gen-binding protein activity in testicular cytosols, epididymal sperm motility, and oocyte fertilization rate, oocyte cleavage rate, and blastocyst development rate after in vitro fertilization (IVF) trials were significantly smaller in group A compared with groups B and C. In contrast, fertilization rate, cleavage rate, and blastocyst development rate after intracytoplasmic sperm injection (ICSI) procedures were not significantly different among groups A, B, C, and D. Both after IVF trials and ICSI techniques, the proportion of the alive offspring to the number of transferred oocytes was significantly smaller in group A than in groups B and C. Cigarette smoke-exposure results in a secretory deficiency of Leydig and Sertoli cells leading to an impaired epididymal sperm maturation process and diminished capacity of spermatozoa to penetrate oocytes. In addition paternal cigarette smoke exposure affects the embryonic ability for implantation.

Effects of paternal cigarette smoking on testicular function, sperm fertilizing capacity, embryonic development, and blastocyst capacity for implantation in rats.

We evaluated the effects of paternal smoking on testicular function, sperm fertilizing capacity, embryonic development, and blastocyst capacity for implantation. Rats of group A were exposed to cigarette smoke for 10 weeks. Rats of group B were exposed to the smoke of incense sticks for 10 weeks. Rats of group C served as a control group. Rats of group D were exposed to cigarette smoke for 7 weeks only. Experimental period was 10 weeks in all groups. At the end of the experimental period serum testosterone responses to human chorionic gonadotropin stimulation, androgen-binding protein activity in testicular cytosols, epididymal sperm motility, and oocyte fertilization rate, oocyte cleavage rate, and blastocyst development rate after in vitro fertilization (IVF) trials were significantly smaller in group A compared with groups B and C. In contrast, fertilization rate, cleavage rate, and blastocyst development rate after intracytoplasmic sperm injection (ICSI) procedures were not significantly different among groups A, B, C, and D. Both after IVF trials and ICSI techniques, the proportion of the alive offspring to the number of transferred oocytes was significantly smaller in group A than in groups B and C. Cigarette smoke-exposure results in a secretory deficiency of Leydig and Sertoli cells leading to an impaired epididymal sperm maturation process and diminished capacity of spermatozoa to penetrate oocytes. In addition paternal cigarette smoke exposure affects the embryonic ability for implantation.

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

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

Increased frequency of female partner chromosomal abnormalities in patients with high-order implantation failure after in vitro fertilization

Objective: To find the type and frequency of chromosomal abnormalities in a selected group of high-order implantation failure (≥6 IVF trials and ≥15 transferred embryos) and to evaluate its impact on pregnancy outcome. Design: A retrospective study. Setting: In vitro fertilization (IVF) unit in a university affiliated hospital. Patient(s): Sixty-five couples with high-order implantation failure in IVF and embryo transfer. Intervention(s): In vitro fertilization/embryo transfer (ET), work-up for implantation failure, cytogenetic analysis of the couple. Main Outcome Measure(s): We studied the type and frequency of chromosomal changes, quality of embryos, cumulative pregnancy rates, and pregnancy outcome. Result(s): The mean number of treatment cycles per patient, before karyotyping was 7.8 ± 2.4 (range: 6 to 16 cycles). The mean cumulative number of all transferred embryos per patient was 25.7 ± 10.3 (range: 9 to 65 embryos). Chromosomal abnormalities were found in 10 of 65 (15.4%) cases: translocations in six, mosaicism in two, and inversion or deletion in another two. The morphologic characteristics of the transferred embryos and the cumulative pregnancy rates were similar in patients with implantation failure with and without chromosomal changes. Three of the 16 patients with abnormal karyotype delivered and three miscarried within a follow-up period of 1 year. Conclusion(s): A high frequency of chromosomal aberrations was found in a selected group of high-order implantation failures, a similar frequency to recurrent miscarriages. Karyotyping is recommended as part of the work-up for repeated implantation failure in assisted reproduction. Treatment options include further IVF trials, preimplantation genetic diagnosis, or oocyte donation, tailored according to the type of chromosomal change. An international registry should be considered to assist in counseling these patients.

Does zona pellucida thickness influence the fertilization rate?

In human in-vitro fertilization (IVF), the cumulus oophorus is routinely removed to assess fertilization and hence the thickness of the zona pellucida is measurable. This study aimed to measure the thickness of the zona pellucida and to assess its influence on fertilization rate in IVF programmes. The zona pellucida thickness varies from 10 to 31 microns with a mean of 17.5 microns. One-way analysis of variance revealed that in IVF trials performed with normal semen, the zona pellucida of fertilized oocytes (16.6 +/- 3.2 microns) was significantly thinner than the zona pellucida of unfertilized oocytes (18.9 +/- 4.0 microns; P < 0.001). As measured on micro-injected oocytes, the zona pellucida thickness did not change between ovulation and 16-20 h after fertilization. Zona pellucida thickness was not related to ooplasm diameter. In conclusion, zona pellucida thickness appears to be an additional factor that should be taken into account when interpreting the fertilization rate. Zona pellucida thickness influences sperm penetration, even when the spermatozoa are considered normal. From a clinical point of view, a thick zona pellucida (> or = 22 microns) could be an indicator for the use of micro-injection procedures.

Fertilization and early embryology: The prognostic value of successful in-vitro fertilization in subsequent trials

A retrospective study comparing in-vitro fertilization (IVF) results in patients with previous successful trials versus new IVF patients was designed with the objective of evaluating the prognosis in both groups. Patients were divided into two groups: group A, 22 patients with previous conception in IVF trials; and group B, 235 new IVF patients. All patients had tubal factor as the only cause of infertility and were stimulated by a standard protocol of gonadotrophin-releasing hormone agonist and human menopausal gonadotrophin. The pregnancy rate in group A (64%) was significantly higher than in group B (19%). In group A, 77% of the patients had three or more grade 1 embryos per transfer as compared with 16% in group B. In group A, 95.5% of patients had easy embryo transfer as compared with 61.2% in group B. The results of the study demonstrated that patients with previous IVF pregnancies have a significantly higher pregnancy rate compared with the control group. The improved pregnancy rate is due to the higher quality of embryos and the higher percentage of easy embryo transfers.

Are there predictive criteria of complicated ovarian hyperstimulation in IVF?

Among 599 trials of in-vitro fertilization (IVF) treatment, complicated ovarian hyperstimulation (OHSS) was diagnosed in 14 cases (2.5%) on the basis of heavy abdominal discomfort and echographic findings (ascites, ovarian enlargement with cysts). Among eight hospitalized patients, four presented with a haemoconcentration and/or electrolytic disturbances. OHSS cases were compared with two control groups for a series of criteria: age, aetiology of infertility, total dose of human menopausal gonadotrophin (HMG), day of oocyte collection, oestradiol (E2) peak level, rate of E2 increase, number of oocytes, number of embryos transferred and embryonic vitality scores. Comparison with a random group of normal IVF trials showed a significant difference for the following parameters: E2 peak level and rate of increase, E2/dose of HMG, E2/day of egg collection and number of oocytes. When OHSS cases were compared to another control group consisting only of high E2 responders (peak E2 greater than 2700 pg/ml), no significant difference was found for any of the above-mentioned criteria. In view of this lack of predictive power of individual criteria, stepwise discriminant analysis was applied, showing that this method might provide a predictive mathematical function for evaluating the risk of OHSS before human chorionic gonadotrophin (HCG) administration. Such a formula, however, should be validated by a multicentric study in which a greater number of OHSS cases would be tested.

Fertility prognosis in IVF treatment of patients with cancelled cycles.

Among 429 patients admitted in our in-vitro fertilization (IVF) programme during the last 3 years, 106 experienced cancellation of one or more treatment cycles. A low oestrogen (E2) response represented by far the main reason for cancellation (144/184). Significantly lower clinical pregnancy rates were found in the group of patients with low E2-cancelled cycles (E2CC) as compared to the rest of the IVF population (13 versus 37%). Further comparison between these groups showed only little or no difference in the mean age of patients, basal FSH levels, number of egg collections, rate of oocytes obtained and fertilized per trial and mean embryonic vitality score. A large majority of E2CC patients were found to belong to the tubal infertility group (75%) as opposed to a lower proportion of tubal cases among other IVF patients (56%). E2CC tubal patients showed a lower pregnancy rate than other cases with abandoned cycles and also than other tubal patients (8.9 versus 25 and 35%, respectively). Mean numbers of previous laparotomies, of attempts at oocyte retrieval and of oocytes recovered per trial were similar in tubal patients with or without E2CC. However, in the E2CC tubal group mean age and basal FSH levels were somewhat higher, whereas E2 peak values and fertilization rates were lower, leading to a greater proportion of trials without embryo transfer. Embryonic scores and the E2/P ratio at day 3 of the luteal phase did not differ significantly. It may be concluded that cancelling of a cycle because of a low E2 response in a patient with tubal infertility is indicative of a poor prognosis in further IVF trials.

Embryo scoring as a prognostic tool in IVF treatment.

A semi-quantitative and non-invasive method for scoring embryos obtained after in-vitro fertilization (IVF) has been defined, aiming at selection of embryos before transfer and at prognostic evaluation of IVF trials. Grading of embryos observed on the inverted microscope was essentially based on the amount of anucleate fragments expelled during early cleavage and on developmental speed. Embryos endowed with a high score were more often associated with pregnancy and in particular with the occurrence of multiple pregnancy. No difference was observed between scores attributed to embryos related to ongoing, aborted or chemical pregnancies. Average embryonic scores corresponding to double and triple transfers differed significantly in failures as well as pregnancies. The better quality of embryos replaced in triple transfers was also apparent from the significantly higher implantation rate per embryo observed in this group. From our results, five criteria including clinical data and embryonic scores can be derived for defining a high risk of multiple pregnancy prior to transfer. It might be warranted to replace only two embryos when these conditions are fulfilled.