IVF-embryo Transfer Programme Research Articles

107 FACTORS THAT INFLUENCE FERTILITY IN AN IVF EMBRYO TRANSFER PROGRAM IN DAIRY HEIFERS

A retrospective study was performed to evaluate factors that influence pregnancy per embryo transfer (P/ET) in an IVF-embryo transfer program. A total of 5026 fresh in vitro-produced embryos were transferred during 2014 and evaluated for effects of embryo quality, embryo stage, size of corpus luteum (CL; 18–19.9 mm or ≥20 mm), interval from GnRH to embryo transfer, number of previous embryo transfer (0, 1, 2, 3, ≥4); and interaction of embryo stage and interval from GnRH to embryo transfer. One group (n = 850) had detection of oestrus after prostaglandin F2α application but most heifers (n = 4176) received fixed timed embryo transfer after a 5-day CIDR-Synch protocol: Day –8 CIDR inserted; Day –3 CIDR removed and prostaglandin F2α; Day –2 prostaglandin F2α; Day 0 GnRH. Ultrasound was performed on Day 6 after GnRH or oestrus to measure CL size and on Day 32 and 60 to determine pregnancy. Data for P/ET were analysed by logistic regression (LOGISTIC procedure, SAS 9.4). Embryo quality influenced P/ET at Day 32 [Grade 1 48.4% (1273/2631) v. Grade 2 37.6% (900/2395); P < 0.01] and at Day 60 [Grade 1 38.9% (1023/2631) v. Grade 2 29.0% (694/2395); P < 0.01], and altered pregnancy loss [Grade 1 19.6% (250/1273) v. Grade 2 22.9% (206/900); P = 0.03]. Stage of the embryo also had an effect on P/ET at Day 32 [Stage 6 35.5%a (582/1641), Stage 7 46.3%b (1431/3092), and Stage 8 54.6%c (160/293); P < 0.01] and at Day 60 [Stage 6 28.2%a (462/1641), Stage 7 36.6%b (1131/3092), and Stage 8 41.6%b (122/293); P < 0.01], but did not affect pregnancy loss (P = 0.22). Interestingly, interval from GnRH (or oestrus) until embryo transfer did not affect P/ET at Day 32 (P = 0.10), 60 (P = 0.23), or pregnancy loss (P = 0.3), nor was there an interaction between interval and embryo stage at Day 32 (P = 0.77), 60 (P = 0.96) or pregnancy loss (P = 0.55). As shown in Table 1, embryo stage 6 was always the lowest and stage 8 always the greatest P/ET regardless of interval from GnRH to embryo transfer. Size of CL also did not affect P/ET at Day 32 (P = 0.09), 60 (P = 0.21), or pregnancy loss (P = 0.90). Number of previous embryo transfer also did not alter P/ET at Day 32 [0 = 43.3% (886/2046), 1 = 44.1% (639/1450), 2 = 43.4% (444/1024), 3 = 42.6% (146/343), and ≥4 = 35.6% (58/163); P = 0.33] or 60 (P = 0.51) or pregnancy loss (P = 0.12). In conclusion, embryo stage and quality are the major factors that impacted P/ET in this study, with surprisingly little effect of interval from GnRH to embryo transfer, size of the CL, and number of previous embryo transfer. Thus, recipient programs for IVF-embryo transfer can be designed with substantial flexibility. Table 1.Effect of embryo stage and recipient synchrony on pregnancies per embryo transfer on Day 32 in recipient dairy heifers

144 PREGNANCY RATES FOLLOWING THE TRANSFER OF IN VITRO FERTILIZED EMBRYOS TO RECIPIENTS ON DAY 6, 7, OR 8 AFTER OESTRUS

The aim of this experiment was to investigate the effect of day of synchrony on the pregnancy rate of recipients following the transfer of Day 7 IVF embryos. In addition, the effect of IVF embryo grade and corpus luteum (CL) grade of recipients was determined. A total of 317 cumulus-oocyte complexes collected from 24 dry Brahman cows by TVR were matured, fertilized, and cultured under standard in vitro production procedures (Fry et al. 2003 Theriogenology 59, 446). A total of 89 (44 Grade 1, 43 Grade 2, and 2 Grade 3, IETS classification) in vitro-produced embryos were transferred to parous 4- to 9-year-old dry Brahman cross recipient cattle 7 days after IVF. Two groups of recipient cows were synchronised one day apart with an 8-day CIDR/pg protocol so that oestrous would be concentrated over 3 days with the middle day aligning with the day of IVF (Day 0). Donors that produced a large number of IVF embryos had these divided and transferred into recipients either on Day –1 or Day +1 of synchrony, and those producing less than 4 IVF embryos were transferred into recipients on Day 0. At embryo transfer the ovaries of the recipient were palpated and then scanned by rectal ultrasound and the grade of CL noted (Grade 1 = large distinct CL by palpation, Grade 2 = small distinct CL by palpation, Grade 3 = CL not distinguishable by palpation). Pregnancy was diagnosed by ultrasound scanning on Day 92. Although recipient numbers were low, differences in pregnancy rate between groups were analysed by Chi-squared. Data from the 2 Grade 3 embryos transferred were not included in the analysis (0/2 pregnant). Similar (P > 0.05) pregnancy rates were found when Day 7 IVF embryos were transferred to either Day 6 (17/32 = 53%), Day 7 (9/24 = 38%), or Day 8 (14/31 = 45%) recipients. Furthermore, neither the grade of the embryo (Grade 1: 20/44 = 45%, Grade 2: 20/43 = 47%) nor the grade of recipient CL (Grade 1: 17/45 = 38%, Grade 2: 17/29 = 59%, Grade 3: 6/13 = 46%) effected pregnancy rate (P > 0.05). This experiment demonstrates the flexibility of the IVF embryo to achieve an acceptable pregnancy rate over a range of recipient stages thereby allowing a high usage rate of good-quality recipients in an IVF embryo transfer program.

3-dimensional endometrial volume as a predictor of pregnancy in in-vitro fertilisation cycles over 2-dimensional endometrial thickness

OBJECTIVE: To evaluate the role of endometrial volume as predictor of pregnancy in In Vitro Fertilisation Cycles (IVF) over endometrial thickness. DESIGN: Prospective, non randomised clinical study. MATERIALS AND METHODS: This was a prospective observational study. A total of 100 infertile women were recruited from our IVF-Embryo transfer program from Feb to March 2014. Endometrial volume was measured on the day of hCG using the VOCAL (Virtual Organ Computeraided AnaLysis) imaging program integrated into Volusen E8 ultrasound system. RESULTS: The mean age was 31.5 years and the mean duration of infertility was 6 years. The mean endometrial thickness was 9.7 mm and the mean endometrial volumewas 4.6 cm3. Overall 31(31%) patients conceived and in these women the endometrial volumewas between 3-8 cm3. All patients who conceived had thickness more than 7 mm. 38 (38%) patients had thickness >7 mm but endometrial volume< 4 cm3 and did not conceive. The positive predictive value of endometrial volume for conception was 40% with sensitivity as high as 93.5%. Endometrial thickness had a high negative predictive value of 68.3% with 97% specificity. Among those who conceived 16 (51%) patients had blood flow in Zone 3, 14 (45%) patients had flow in Zone 2 and only one had flow in Zone 1. CONCLUSION: With a thin endometrium and low endometrial volume, the probability of conception is very low in an in-vitro fertilisation/ embryo transfer cycle and cryopreservation should be recommended. Endometrial thickness more than 7 mm and volume more than 3cm3 together increase the probability of pregnancymore than thickness alone as volume is an objective measure of implantation potential of endometrium. However furthur study is needed for definitive conclusion.

Does supplementation of in-vitro culture medium with melatonin improve IVF outcome in PCOS?

Human pre-ovulatory follicular fluid (FF) contains a higher concentration of melatonin than serum. The aim of this study was to evaluate the effect of melatonin supplementation of culture medium on the clinical outcomes of an in-vitro maturation (IVM) IVF-embryo transfer programme for patients with polycystic ovarian syndrome (PCOS). Melatonin concentrations in the culture media of granulosa cells (GC) or cumulus-oocyte-complexes (COC) were measured and the clinical outcomes after using IVM media with or without melatonin were analysed. In the culture media of GC or COC, melatonin concentrations gradually increased. When human chorionic gonadotrophin priming protocols were used, implantation rates in the melatonin-supplemented group were higher than those of the non-supplemented control group (P<0.05). Pregnancy rates were also higher, although not significantly. The findings suggest that the addition of melatonin to IVM media may improve the cytoplasmic maturation of human immature oocytes and subsequent clinical outcomes. It is speculated that follicular melatonin may be released from luteinizing GC during late folliculogenesis and that melatonin supplementation may be used to improve the clinical outcomes of IVM IVF-embryo transfer.Melatonin is primarily produced by the pineal gland and regulates a variety of important central and peripheral actions related to circadian rhythms and reproduction. Interestingly, human pre-ovulatory follicular fluid contains a higher concentration of melatonin than serum. However, in contrast to animal studies, the direct role of melatonin on oocyte maturation in the human system has not yet been investigated. So, the aim of the study was to evaluate the effect of melatonin supplementation of culture medium on the clinical outcome of an in-vitro maturation (IVM) IVF-embryo transfer programme for PCOS patients. The melatonin concentrations in culture medium of granulosa cells (GC) or cumulus-oocyte-complexes (COC) were measured and the clinical outcomes of IVM IVF-embryo transfer using IVM medium alone or supplemented with melatonin were analysed. In the culture media of GC or COC, the melatonin concentration gradually increased. With human chorionic gonadotrophin priming, the pregnancy and implantation rates in the melatonin-supplemented group were higher than those of the non-supplemented control (P<0.05). Our findings suggest that follicular melatonin is released from luteinizing GC during late folliculogenesis and plays a positive role in oocyte maturation. Therefore, addition of melatonin into IVM medium may improve cytoplasmic maturation of human immature oocytes and subsequent clinical outcomes.

157 PREGNANCIES AND CALVES AFTER TRANSFER OF IN VITRO-PRODUCED RIVER BUFFALO EMBRYOS AFTER CRYOPRESERVATION

In the buffalo, the use of embryo-based biotechnologies for breeding and genetic improvement is still very limited because multiple-ovulation embryo transfer delivers poor results compared with cattle and in vitro embryo production has been used mainly for research purposes. At present, very few reports are available on the transfer of in vitro-produced (IVP) and cryopreserved buffalo embryos. Therefore, the scope of this work was to perform a pilot study to evaluate the viability of frozen-thawed IVP embryos by nonsurgical embryo transfer to recipients in an IVF-embryo transfer program on a farm located on the north coast of Colombia, South America. Buffalo oocytes were recovered at the slaughterhouse from selected donors, matured in vitro for 18 to 20 h in TCM-199 + 10% FCS and 0.5 IU of FSH and 0.5 IU of LH in 5% CO2 at 38.5°C. Four different bulls were used for IVF. After thawing, the semen was separated on a Percoll® gradient and then diluted into SOF-IVF media supplemented with 1 μg mL–1 of heparin and phenylalanine. Presumptive zygotes were cultured in modified SOF supplemented with MEM amino acids for 6 days. Half of the medium was replaced on Day 4 and 6. Developing embryos were selected for freezing on Day 6 and 7. Grade 1 embryos were frozen at the blastocyst stage by slow cooling in 10% glycerol or 1.5 M ethylene glycol. Recipients (heifers n = 79 and uniparous cows n = 17) were synchronized using the CIDR-Synch protocol: on Day 0, gonadotropin-releasing hormone was injected and a CIDR was inserted; on Day 7, prostaglandin F2α was administered; on Day 9, the CIDR was removed; on Day 11, a second injection of gonadotropin-releasing hormone was given; and on Day 17, the embryo was transferred. Each female received, nonsurgically, 1 or 2 embryos in the ipsilateral horn to the functional corpus luteum evaluated by ultrasonography. Pregnancies were evaluated by ultrasonography 30 days after transfer and confirmed by rectal palpation 30 days later. This work was performed in 2 successive experiments during the breeding seasons (January and December, respectively). Overall, 96 recipients were transferred, with 136 embryos obtaining 23 pregnancies (24.2%). There were no statistical differences in pregnancy rate between heifers and cows (25.3 vs 17.7%) and between single (n = 56) and double (n = 39) embryo transfers (21.4 vs 27.5%) by chi square test (P &gt; 0.05). To date, 4 females and 5 males have been born by spontaneous calving (1 stillborn male due to dystocia), 3 pregnancies have been aborted (13%) and 11 pregnancies are ongoing (&gt;7 months). The pregnancy rate obtained in this study in farm conditions (24.2%) is lower than generally obtained with frozen IVP cattle embryos, but it is still a good result in buffalo, where even conventional AI provides a lower success rate as compared with cattle. Finally, this work demonstrates that in vitro embryo production can be successfully implemented in buffalo breeding programs for the exploitation of superior genetics. This work was supported by Regione Lombardia, Por Fers 2007–2013, n°13827741, InnovaB.

Identification and validation of candidate biomarkers involved in human ovarian autoimmunity

Antibodies to multiple ovarian antigens have been proposed as markers of ovarian autoimmunity. The role of ovarian autoantibodies has been widely discussed in the pathophysiology of premature ovarian failure and unexplained infertility, but the autoantigens are yet to be identified. Three immunodominant ovarian autoantigens, α-actinin 4 (αACTN4), heat shock 70 protein 5 (HSPA5) and β-actin (ACTB), have been identified using anti-ovarian antibody-positive sera from women with idiopathic premature ovarian failure ( n = 50) and women undergoing IVF ( n = 695), using mass spectrometry. These autoantigens were subsequently validated using Western blot, immunohistochemistry and enzyme-linked immunosorbent assay. These autoantigens are localized to different components of the ovary such as the ooplasm of the oocyte, theca, granulosa, corpus luteum and zona pellucida. All the above antigens were found to be expressed in the ooplasm throughout follicular development. All the autoantigens are expressed specifically in the oocyte except αACTN4. The three autoantigens could contribute to the array of biomarkers to be used for developing specific and sensitive tests for diagnosis of women at risk of premature ovarian failure and IVF failure due to ovarian autoimmunity and could give an insight into the molecular mechanisms involved in the pathophysiology of these conditions. Anti-ovarian antibodies (AOA) have been reported in women with premature ovarian failure and unexplained infertility. Among women with infertility, those with evidence of ovarian autoimmunity appear to have poorer IVF–embryo transfer outcomes. Diagnosis of an autoimmune mechanism in these pathologies has relied for a long time on the detection of AOA. Little is known about the precise ovarian antigenic targets in terms of its molecular and cellular identities that are recognized by the antibodies and immune cells in autoimmune diseases of the ovary. In the present study, we observed that 31% of the total women recruited under an IVF–embryo transfer programme (group I) and 46% of women with premature ovarian failure (group II) tested positive for AOA. Three immunodominant ovarian autoantigens, namely non-muscle α-actinin 4, heat shock 70 protein 5 and cytoplasmic β-actin, were identified using mass spectrometry and validated and characterized using AOA-positive sera from women from both groups. Further investigation of the identified targets could give us an insight into the molecular mechanism involved in the pathophysiology of human ovarian autoimmunity.

Establishment of human embryonic stem cell lines from frozen-thawed blastocysts using STO cell feeder layers

Recently, human embryonic stem (hES) cells have become very important resources for basic research on cell replacement therapy and other medical applications. The purpose of this study was to test whether pluripotent hES cell lines could be successfully derived from frozen-thawed embryos that were destined to be discarded after 5 years in a routine human IVF-embryo transfer programme and whether an STO cell feeder layer can be used for the culture of hES cells. Donated frozen embryos (blastocysts or pronuclear) were thawed, and recovered or in vitro developed blastocysts were immunosurgically treated. All inner cell masses were cultured continuously on an STO cell feeder layer and then presumed hES cell colonies were characterized. Seven and two cell lines were established from frozen-thawed blastocysts (7/20, 35.0%) and pronuclear stage embryos (2/20, 10.0%), respectively. The doubling time of hES cells on the immortal STO cell feeder layer was approximately 36 h, similar to that of cells grown using fresh mouse embryonic fibroblast (MEF) feeder conditions. Subcultured hES cell colonies showed strong positive immunostaining for alkaline phosphatase, stage-specific embryonic antigen-4 (SSEA-4) and tumour rejection antigen 1-60 (TRA1-60) cell surface markers. Also, the hES colonies retained normal karyotypes and Oct-4 expression in prolonged subculture. When in vitro differentiation of hES cells was induced by retinoic acid, three embryonic germ layer cells were identified by RT-PCR or indirect immunocytochemistry. This study indicates that establishment of hES cells from frozen-thawed blastocysts minimizes the ethical problem associated with the use of human embryos in research and that the STO cell feeder layer can be used for the culture of hES cells.

Role of utero–ovarian vascular impedance: predictor of ongoing pregnancy in an IVF–embryo transfer programme

The objective of this study was to evaluate the role of uterine, endometrial and follicular blood flow in prediction of ongoing pregnancy after assisted conception. A prospective observational study was conducted on 53 women undergoing IVF treatment. Transvaginal colour and pulsed Doppler measurements were performed on the day that pituitary suppression was confirmed, on day 10 of ovarian stimulation and on the day prior to human chorionic gonadotrophin injection. On the last day of ovarian stimulation, blood flow in the ascending uterine artery of the women who would conceive was characterized by significantly lower pulsatility index values. Sub-endometrial vascular impedance was comparable in the pregnant and non-pregnant groups. There were no differences in the perifollicular vascularity between pregnant and non-pregnant women. The chance of achieving pregnancy predicted by uterine artery Doppler and perifollicular blood flow in women whose PI values were higher than 3.26 and 1.08 was very low, with a sensitivity of 1.00 and specificity of 0.59 and 0.82 respectively. The data provide evidence for an association between utero–ovarian perfusion and reproductive outcome following IVF treatment. Uterine and ovarian vascular impedance values identify those women whose pregnancy chances are significantly limited. Measures to decrease vascular impedance in such women might enhance pregnancy rates by improving embryo quality and uterine receptivity for implantation.

The significance of cytokines, chemical composition, and murine embryo development in hydrosalpinx fluid for predicting the IVF outcome in women with hydrosalpinx.

The aim of this study was to determine whether differences in cytokine concentrations, chemical composition, and murine embryogenesis in hydrosalpinx fluid could be observed between women with hydrosalpinx who achieved and did not achieve pregnancy. Hydrosalpinx fluids were collected from 39 infertile women participating in an IVF-embryo transfer programme at a university teaching hospital. The fluids were analysed for concentrations of cytokine concentration (epidermal growth factor, interferon-gamma, leukaemia inhibitory factor, and tumour necrosis factor-alpha) and chemical composition. Mouse embryos were cultured in medium with various concentrations (0, 10, 50, 100%) of hydrosalpinx fluid. Logistic regression demonstrated that a murine blastocyst formation rate >or=53.3% in 50% hydrosalpinx fluid (OR = 16.6, 95% CI 2.4-116.1, P = 0.005) and patient age (OR = 0.778, 95% CI 0.61-0.99, P = 0.045) were independent predictors of IVF outcome. The diagnostic accuracy did not significantly improve when age and number of good quality embryos transferred was taken into account. The chemical composition and cytokine concentrations of hydrosalpinx fluid were not predictive of subsequent IVF outcome. The mouse embryo assay of hydrosalpinx fluid might potentially serve as a predictor of subsequent IVF outcome in women with hydrosalpinx. This technique may be useful in selecting appropriate treatment options for patients with hydrosalpinx undergoing IVF treatment.

Transvaginal ultrasound-guided embryo transfer improves pregnancy and implantation rates after IVF.

Attempts are constantly being made to improve clinical pregnancy rates after IVF and embryo transfer. Since November 1998, we have gradually been adopting transvaginal ultrasound guidance during embryo transfer. We retrospectively examined the efficacy of this method on pregnancy and implantation rates. The results of 846 cycles from our IVF-embryo transfer programme were analysed and comparisons were made between those carried out using ultrasound guidance and those by the clinical touch method. Higher pregnancy and implantation rates (28.9 and 15.2% respectively) were found in the group using the transvaginal ultrasound guidance during embryo transfer compared with those in the group using the clinical touch method (13.1 and 7.0% respectively). The differences were statistically significant (P < 0.01). There was no significant difference in ectopic pregnancy rates between the two groups. The use of transvaginal ultrasound-guided embryo transfer significantly improved both pregnancy and implantation rates. Although technically difficult, we suggest its use may maximize the chances of achieving a successful pregnancy outcome.

Three-dimensional endometrial volume estimation as a predictor of pituitary down-regulation in an IVF-embryo transfer programme.

To examine the role of three-dimensional (3D) volume estimation in predicting pituitary down-regulation in an IVF-embryo transfer programme, 46 women were treated with buserelin acetate for down-regulation prior to and concomitantly with ovarian stimulation. Receiver operating characteristic (ROC) curve analysis was used for statistics. The area under the curve (AUC) provides a measure to show how good (AUC close to 1) or how poor (AUC close to 0.5) a test is. If down-regulation was defined as oestradiol <40, <60 or <80 pg/ml, the AUC of 3D-ultrasound was 0.57, 0.83 and 0.97 respectively. The highest sensitivity (100%) and specificity (93.2%) of 3D-ultrasound was achieved if down-regulation was defined as oestradiol <80 pg/ml (n = 44), with an endometrial volume of 1.9 ml. If down-regulation was defined as oestradiol <60 pg/ml (n = 42) the best combination of highest sensitivity (95.2%) and specificity (75. 0%) was obtained with an endometrial volume of 1.9 ml. The comparison of 3D volume calculation with endometrial thickness measurement revealed no significant difference in predicting down-regulation. 3D volume estimation provides a new tool for diagnosing relative hypo-oestrogenism or down-regulation, but it was not superior to two-dimensional ultrasound.

Increased prevalence of thyroid antibodies in euthyroid women with a history of recurrent in-vitro fertilization failure.

This study was undertaken to evaluate whether the presence of thyroid antibodies in euthyroid women is associated with an adverse outcome in an in-vitro fertilization (IVF)-embryo transfer programme. In 24 women (study group: mean age +/- SD: 31.5 +/- 4.4 years) who failed to conceive after having three or more cycles of IVF and embryo transfer, serum concentrations of thyroglobulin (TG), thyroid peroxidase antibodies (TPO) and anticardiolipin antibodies (IgG and IgM) were measured using commercially available kits. The control group comprised 24 consecutive patients without endocrine dysfunction (mean age +/- SD: 30.3 +/- 4.1 years) seeking infertility treatment in our department of assisted reproduction. All patients in both the study and the control groups were determined to be euthyroid by demonstrating normal concentrations of thyroid-stimulating hormone (TSH). In the study and control groups respectively, 13 and two patients demonstrated positive titres of TG, TPO or both thyroid antibodies (Fisher's exact test: P = 0.002). Mean serum concentrations of TG were significantly increased in the study group compared to the control subjects (156 +/- 167 IU/ml versus 33.5 +/- 32.0 IU/ml; U-test: P = 0.009). Serum concentrations of TPO and anticardiolipin antibodies were similar in both groups. Our investigations revealed that thyroid antibodies might be independent markers for reproductive failure in an IVF-embryo transfer programme.

Dexamethasone supplementation to gonadotropin stimulation for in vitro fertilization in polycystic ovarian disease.

This study was conducted to determine whether glucocorticoid supplementation for patients with polycystic ovarian disease during ovulation induction with gonadotropins for in vitro fertilization (IVF) therapy is beneficial. Seventy-one cycles of patients undergoing first attempts at IVF, with classical polycystic ovarian disease and hyperandrogenemia, who enrolled in the IVF-embryo transfer program, were evaluated retrospectively. In 20 cycles (20 patients) glucocorticoid supplementation was noted and compared to 51 cycles (51 patients) without glucocorticoid as adrenal androgen suppression. Ovaries were stimulated by gonadotropin releasing hormone agonist, human menopausal gonadotropin, and dexamethasone. Ovarian responsiveness and IVF-embryo transfer outcome were analyzed and included the number of follicles > 17 mm in diameter, serum estradiol concentration on the day of human chorionic gonadotropin administration, number of human chorionic gonadotropin ampoules administered, number of oocytes retrieved, percentage of oocytes fertilized, number of embryos transferred, implantation rate, and number of clinical pregnancies and their outcome. The results showed that the pregnancy rate in patients who received glucocorticoid was 22.1%, compared to 26% in the controls (statistically insignificant). The IVF cycle variables studied revealed no statistically significant differences. Our observations did not support the notion that adrenal androgen suppression by glucocorticoid, or as an adjuvant therapy, is beneficial to patients with polycystic ovarian disease who enrolled in an IVF-embryo transfer program.

Detection of reactive oxygen species (ROS) and apoptosis in human fragmented embryos.

In human in-vitro fertilization (IVF)-embryo transfer, the in-vitro culture environment differs from in-vivo conditions in that the oxygen concentration is higher, and in such conditions the mouse embryos show a higher concentration of reactive oxygen species (ROS) in simple culture media. ROS are believed to cause damage to cell membranes and DNA fragmentation in somatic cells. This study was conducted to ascertain the level of H2O2 concentration within embryos and the morphological features of cell damage induced by H2O2. A total of 62 human oocytes and embryos (31 fragmented, 15 non-fragmented embryos, 16 unfertilized oocytes) was obtained from the IVF-embryo transfer programme. The relative intensity of H2O2 concentrations within embryos was measured using 2',7'-dichlorodihydrofluorescein diacetate by Quanti cell 500 fluorescence imaging and DNA fragmentation was observed with transmission electron microscopy and an in-situ apoptosis detection kit. The H2O2 concentrations were significantly higher in fragmented embryos (72.21 +/- 9.62, mean +/- SEM) compared to non-fragmented embryos (31.30 +/- 3.50, P < 0.05) and unfertilized oocytes (30.75 +/- 2.67, P < 0.05). Apoptosis was observed only in fragmented embryos, and was absent in non-fragmented embryos. Electron microscopic findings confirmed apoptotic bodies and cytoplasmic condensation in the fragmented blastomeres. We conclude that there is a direct relationship between increased H2O2 concentration and apoptosis, and that further studies should be undertaken to confirm these findings.

The outcome of an in vitro fertilization program in women with polycystic ovary syndrome

The objective of this study was to examine the efficacy of D-triptorelin in the long protocol and follicle-stimulating hormone (FSH) in an IVF-embryo transfer program in a group of patients with polycystic ovary syndrome (PCOS) who had earlier failed to respond or did not conceive after clomiphene citrate (CC)/human menopausal gonadotropin (hMG) or flare up' gonadotropin-releasing hormone agonist (GnRHa)ZhMG stimulation.Eighty-nine women with PCOS (based on typical ultrasound criteria) had 1-3 treatment cycles without success. The stimulation protocol was changed to D-triptorelin given in midluteal phase and when pituitary desensitization was achieved, FSH administration was started. The clinical pregnancy rate per transfer in this ‘negatively selected’ group of PCOS patients was 29%. This was the same as the rate for a group of women with tubal factor, as was the spontaneous miscarriage rate.Although the use of GnRH agonists in the long protocol increases the costs of treatment, the number of cancelled cycles is reduced. The pregnancy rate increased in this group of women with PCOS.

The embryo versus endometrium controversy revisited as it relates to predicting pregnancy outcome in in-vitro fertilization-embryo transfer cycles.

To evaluate embryonic and endometrial factors for their value in predicting pregnancy outcome in in-vitro fertilization (IVF) and embryo transfer, a retrospective data collection and prospective uterine artery colour Doppler imaging study was performed in a university-based IVF-embryo transfer programme setting. A total of 210 patients were included and grouped as follows: (I) IVF with controlled ovarian stimulation (214 cycles); (II) frozen-thaw cycle of autologous embryos (30 cycles); (III) oocyte donation, no cryopreservation (12 cycles); (IV) frozen-thaw cycle with embryos from donated oocytes (10 cycles). Embryo quality was significantly better in pregnant than non-pregnant cycles (group I, P = 0.0104; groups II-IV, P = 0.0418). The endometrial echo was significantly thicker in pregnant versus non-pregnant patients in group I (P = 0.0059), but not in groups II-IV (P = 0.741). Past uterine surgery or abnormalities had no effect on pregnancy outcome. There were no significant differences in mean uterine artery resistance index or peak systolic velocity in pregnant versus non-pregnant patients in groups II-IV. Thus, embryo quality is the most reliable predictor of pregnancy outcome. Endometrial measurements were significantly thicker in subsequently pregnant patients only in group I, where the endometrium reflects the hormonal environment. Doppler parameters were not useful in predicting pregnancy outcome.

Migration sedimentation technique as a predictive test for the fertilizing capacity of spermatozoa in an in-vitro fertilization programme.

The migration-sedimentation technique (MST) has been proposed as a means of separating high quality motile spermatozoa. The present study was conducted in order to evaluate whether sperm performance following separation by MST predicts their fertilizing capacity in an in-vitro fertilization (IVF) programme. Ninety semen specimens were analysed for use in an IVF-embryo transfer (ET) programme. Each specimens was divided into two parts: one was processed in the IVF programme and was used after sperm swim-up separation for insemination of human ova. The other aliquot (0.2 ml) was separated by MST, and the sperm then characterized by their concentration, motility, degree of motility and morphology. Sperm characteristics after separation by MST were then correlated with the results of the IVF-fertilization rates. In 79 of 90 IVF-ET cycles, at least one oocyte was fertilized. All post-MST sperm characteristics were significantly higher in cycles with fertilizations compared to IVF cycles without fertilization. A larger percentage of the total motile spermatozoa were recovered after MST in semen specimens with fertilization, compared to semen specimens without fertilization (39.9 +/- 3.6 and 20.6 +/- 6.6%, respectively; P < 0.05). This value was correlated with the percentage of fertilized oocytes (r = 0.24; P < 0.02). More IVF cycles with fertilizations were recorded in cases in which the recovery of motile sperm was > 25% (P < 0.005), or when more than 1.5 x 10(6) motile spermatozoa were recovered after MST (P < 0.0001). As sperm characteristics after MST correlated significantly with their fertilizing capacity, the MST test could be used in evaluation of the fertilizing capacity of spermatozoa.