Mild stimulation approach for In Vitro Fertilization treatment: Retrospective data from one Danish Centre.

AIM OF STUDY: Over the last decade, the laboratory procedures in artificial reproduction have improved. Hyperstimulation causes an overload of eggs which will never be used. The present study was designed to evaluate the efficiency of a mild stimulation. To obtain oocytes for In Vitro Fertilization (IVF) a short antagonist protocol using Tamoxiphene and FSH was compared to conventional IVF. METHODS: A retrospective and observatory study including all patients with unexplained infertility. In total 720 cycles with mild stimulation protocol and 8,446 cycles with regular short antagonist IVF protocol were analysed. The observation period was from January 2011 until September 2017. All patients were recruited in the same time period and allocated to different treatments upon their request. Low stimulation using orally administrated anti-estrogenic drugs combined with FSH in the form of injections was used in order to obtain up to four mature follicles. RESULTS: The clinical pregnancy rate (CPR) per embryo transfer (ET) was 25% for the mild stimulation group. The CPR for the control group with conventional IVF was 23%. CONCLUSION: Mild stimulation may be an important step towards an easier IVF approach, more tolerable for women, easier and cheaper for the women and the society, while maintaining an acceptable success rate in terms of CPR. Large prospective studies need to be performed.

A multi-centre phase 3 study comparing efficacy and safety of Bemfola(®) versus Gonal-f(®) in women undergoing ovarian stimulation for IVF.

Bemfola (follitropin alfa) (Finox AG, Switzerland), a new recombinant FSH, has a comparable pharmacological profile to that of Gonal-f (Merck Serono, Germany), the current standard for ovarian stimulation. A randomized, multi-centre, Phase 3 study in women undergoing IVF or intracytoplasmic sperm injection (n = 372) showed Bemfola yielding similar efficacy and safety profiles to Gonal-f. Women aged 20-38 years of age were randomized 2:1 to receive a single, daily, subcutaneous 150 IU dose of either Bemfola or Gonal-f. This study tested equivalence in the number of retrieved oocytes using a pre-determined clinical equivalence margin of ±2.9 oocytes. Compared with Gonal-f, Bemfola treatment resulted in a statistically equivalent number of retrieved oocytes (Bemfola 10.8 ± 5.11 versus Gonal-f 10.6 ± 6.06, mean difference: 0.27 oocytes, 95% confidence interval: -1.34, 1.32) as well as a similar clinical pregnancy rate per embryo transfer in first and second cycles (Bemfola: 40.2% and 38.5%, respectively; Gonal-f: 48.2% and 27.8%, respectively). No difference in severe ovarian hyperstimulation syndrome was observed between treatment groups (Bemfola: 0.8%; Gonal-f: 0.8%). This study demonstrates similar clinical efficacy and safety profiles between Bemfola and Gonal-f, and suggests that Bemfola can be an appropriate alternative in ovarian stimulation protocols.

Recombinant LH supplementation to recombinant FSH during the final days of controlled ovarian stimulation for in vitro fertilization. A multicentre, prospective, randomized, controlled trial.

BACKGROUND: The purpose of this multicentre, multinational trial was to study whether rLH supplementation to recombinant FSH (rFSH) during the late follicular phase increased pregnancy rates. METHODS: After down-regulation with nafarelin, 526 women were randomized on Day 1 of stimulation to use either rFSH (Gonal-F) alone (n = 261) or to continue after Day 6 of stimulation with both rFSH (Gonal-F) and rLH (Luveris) (n = 265) from Day 6. The starting dose of rFSH was 150-225 IU/day according to age below or above 35 years. RESULTS: Ongoing pregnancy rate at week 10-12 was 28.7% after rFSH alone and 27.2% after rFSH + rLH. This showed no evidence of a difference. Administration of rLH significantly (P< 0.001) increased serum LH. Ongoing pregnancy rates in patients with low LH levels (<33 percentile) on Days 1 and 6 of stimulation showed no difference between the group treated with rFSH only (23.9% low Day 1 LH; 22.1% low Day 6 LH) versus rFSH + rLH (25.0% low Day 1 LH; 28.9% low Day 6 LH). CONCLUSIONS: Supplementing rFSH with daily doses of 75-150 IU of rLH during the second half of the follicular phase showed no evidence of increasing the ongoing pregnancy rates in the general population. (ClinicalTrials.gov, trial number: KF02-035/03).

Spontaneous abortion in IVF couples--a role of male welding exposure.

BACKGROUND: Fume from welding of stainless steel contains hexavalent chromium, which in animal studies can induce paternally mediated spontaneous abortion. Human studies have shown conflicting results. The best studies include early pregnancy experience, but these are expensive to conduct. In vitro fertilization (IVF) provides new design opportunities. Our aim was to study pregnancy survival in IVF treated women with respect to paternal welding exposure. METHODS: We mailed a questionnaire to 5879 couples from the Danish IVF register that covers all IVF treatments after 1993 (response ratio 68.2%). A subgroup of male metal workers received a second questionnaire on exposure to welding (n = 319 men, response ratio 77%). Information on outcome was collected from national health registers. Survival of the first hCG-positive pregnancy was analysed using Cox regression. RESULTS: The proportion of pregnancies terminated by spontaneous abortion before 28 gestational weeks was 18% (n = 91 pregnancies) and 25% (n = 128) in pregnancies with paternal exposure to stainless steel welding and mild steel welding, respectively. In the reference group of 2925 pregnancies the abortion ratio was 28%. The risk ratio for pregnancies with paternal exposure to stainless steel was 0.6 (95% CI 0.4-1.0). CONCLUSIONS: We found no increased risk of spontaneous abortion in IVF treated women, who became pregnant by a man exposed to welding of any sort. Since the process of fertilization and selection of IVF pregnancies differs from natural pregnancies the negative results need not apply to other pregnancies.

Reproductive outcome using a GnRH antagonist (cetrorelix) for luteolysis and follicular synchronization in poor responder IVF/ICSI patients treated with a flexible GnRH antagonist protocol.

To investigate the possible beneficial effect of a new stimulation protocol (termed 'CRASH') on the outcome of poor responder patients, a multicentre, prospective longitudinal study including a total of 36 women undergoing 72 IVF/intracytoplasmic sperm injection (ICSI) cycles with patients serving as their own controls, was conducted. A poor responder patient was defined as a patient with four or fewer oocytes extracted from five or fewer follicles and with a total FSH consumption exceeding 2000 IU in a preceding long agonist down-regulation protocol. The CRASH protocol included 3 mg of the gonadotrophin-releasing hormone (GnRH) antagonist cetrorelix given in the late luteal phase on cycle day 23. Stimulation with recombinant human FSH (rhFSH) started on cycle day 2, followed by a flexible GnRH antagonist protocol. The results showed significantly more follicles (5.4 versus 3.5), oocytes (4.3 versus 2.4) and transferable embryos (1.8 versus 0.8) with the CRASH protocol as compared with the preceding long protocol (P < 0.005 in all cases). The implantation rate and pregnancy rate per transfer was 18.4 and 38.5% respectively, approaching the clinical outcome of normal responder patients. The CRASH protocol thus may constitute an attractive alternative to conventional protocols for low responder patients, improving their clinical outcome.

Assisted hatching improves implantation rates on cryopreserved-thawed embryos. A randomized prospective study.

BACKGROUND: Focus on the hatching process has so far been in the field of fresh embryos. Cryopreserved-thawed embryos have a lower rate of pregnancy than fresh embryos, which might be due to hardening of the zona pellucida. METHODS: During a 2 year period, a prospective randomized study enrolling 253 cryopreserved-thawed cycles was performed on day 2 embryos. Pseudorandomization to assisted hatching or a control group was done on the basis of even and odd dates for thawing. One hour before embryo transfer, hatching was carried out using acidic Tyrode's solution. RESULTS: Among 136 embryos exposed to assisted hatching, 11.4% (30) were implanted compared with only 5.8% (13) of 117 embryos not exposed to assisted hatching (P<0.05, chi(2) test). No difference in the rate of clinical pregnancy and positive serum HCG was observed between the two groups. Very few women >38 years old were included in the study, and no significant difference according to age could be found between the groups. CONCLUSIONS: These results show that assisted hatching using acidic Tyrode's solution increases the implantation rate of cryopreserved-thawed embryos (P<0.05).

Pesticide exposure in male farmers and survival of in vitro fertilized pregnancies.

BACKGROUND: Male-mediated spontaneous abortion is well described among animals, but less well documented in humans. Studies that include early pregnancy experience are expensive to conduct, but IVF provides new opportunities. MATERIALS AND METHODS: The Danish IVF register covers all IVF treatments performed after 1993. We mailed a questionnaire to 5879 women (response rate 68.2%). A subgroup of exposed male partners received a questionnaire on specific exposure to pesticides and growth retardants (n = 128 men, response rate 81.3%). Information on outcome was collected from national health registers. Survival of the first HCG-positive pregnancy was analysed using Cox regression. RESULTS: The proportion of pregnancies terminated by spontaneous abortion before 28 gestational weeks was 19.7% (n = 66 pregnancies), 19.7% (n = 61), 21.3% (n = 47) and 22.2% (n = 18) in pregnancies with paternal exposure to herbicides, fungicides, pesticides and growth retardants. respectively. In the reference group of 2925 pregnancies, the abortion rate was 28.4%. The differences in survival were not statistically significant. CONCLUSIONS: We found no increased risk of spontaneous abortion in IVF-treated women attributable to paternal agricultural application of pesticides and growth retardants. Exposure to potentially harmful pesticides in Denmark is relatively low, and the findings are restricted to countries with similar standards of protection.

Infertility despite surgery for cryptorchidism in childhood can be classified by patients with normal or elevated follicle-stimulating hormone and identified at orchidopexy.

OBJECTIVE: To analyse infertility despite orchidopexy in childhood. PATIENTS AND METHODS: The study comprised patients with cryptorchidism (70 bilateral and 65 unilateral) who had a simultaneous biopsy taken at orchidopexy in childhood, and in adulthood had analyses of semen and FSH. In adulthood 42 formerly bilateral cryptorchid boys had repeat testicular biopsies taken. Infertility was suspected in men with < 5 million sperm/mL in the best sample of semen and concomitant poor sperm motility, and who were classified by follicle-stimulating hormone (FSH) values. At orchidopexy the number of spermatogonia/tubule and the germ cell differentiation were measured. In adulthood the percentage of tubules with complete spermatogenesis, spermatogenic arrest and Sertoli-cell only status was assessed. RESULTS: Infertility was suspected in 38 of 70 (54%) of formerly bilateral and six of 65 (9%) formerly unilateral cryptorchid patients. High FSH values were expected in these suspected infertile patients, but 15 of 38 (59%) formerly bilateral and five of six formerly unilateral cryptorchid patients had normal FSH values. These patients were identified in childhood at orchidopexy; those with bilateral cryptorchidism generally presented with germ cells, but the mean number of spermatogonia per tubule was < 30% of the lowest normal value, and the germ cells were seldom normally differentiated, whereas those with unilateral cryptorchidism generally lacked germ cells in the biopsies. No patients had a decreased FSH value. CONCLUSION: Despite surgery for cryptorchidism, infertility was probable in a third (44 of 135) of the patients. We expected high FSH values in these patients, but in 45% (20/44) the FSH values were normal. These patients may have relative FSH deficiency. At orchidopexy these patients were identified to be bilaterally cryptorchid with few germ cells and those unilaterally cryptorchid had none in the biopsy. After orchidopexy in childhood, additional hormonal treatment, e.g. recombinant FSH or buserelin, may be indicated in these patients.

Incidence of apoptosis in granulosa cells from immature human follicles.

The aim of this study was to investigate the incidence of apoptosis in granulosa cells from immature human follicles undergoing in vitro maturation (IVM) and to compare the incidence of apoptotic granulosa cells (i) between FSH-primed and unprimed normal ovaries and (ii) between polycystic and normal ovaries. Furthermore, the incidence of apoptosis was related to maturation and subsequent fertilization and cleavage of the oocytes from the corresponding ovary. Seventy women undergoing 70 IVM cycles were included. Group 1 consisted of patients with normal ovaries (n = 52) and group 2 consisted of patients with polycystic ovaries (n = 18). Patients in group 1 were subdivided into two groups according to priming with FSH before aspiration. In group 1a (n = 27 cycles) oocytes were obtained in unstimulated cycles. In group 1b (n = 25 cycles) oocytes were obtained after priming with recombinant FSH for 3 days initiated on day 3 after spontaneous menstruation. In group 2 all patients were primed with recombinant FSH for 3 days before aspiration. Aspiration was performed transvaginally and cumulus-enclosed oocytes were matured for 28-30 h before fertilization. Granulosa cells were collected from follicular aspirates. An APOPTAG detection kit was used to stain the granulosa cells and to detect apoptosis. The incidence of apoptosis in granulosa cells was decreased in follicles from FSH-primed normal ovaries compared with follicles from unprimed normal ovaries and FSH-primed polycystic ovaries. No difference was found between granulosa cells from FSH-primed polycystic ovaries and granulosa cells from unstimulated normal ovaries. No differences in maturation rate, fertilization rate, cleavage rate and implantation rate were observed when oocytes from a polycystic ovary were compared with oocytes from an unstimulated normal ovary. In unstimulated cycles, the ovaries were grouped according to the presence of a dominant follicle. The incidence of apoptosis was significantly higher in granulosa cells from an ovary without a dominant follicle compared with granulosa cells from an ovary with a dominant follicle. The rates of maturation, fertilization and cleavage did not differ between the two groups.

Benefit of FSH priming of women with PCOS to the in vitro maturation procedure and the outcome: a randomized prospective study.

The aim of this study was to determine whether the rates of in vitro oocyte maturation, fertilization and cleavage, as well as implantation rate and pregnancy rate, could be improved by low-dose priming with FSH in vivo before retrieval of immature oocytes in patients with polycystic ovary syndrome (PCOS). From March 1998 to June 2000, a total of 28 women underwent 36 completed treatment cycles, randomized sequentially in one of two groups. Women in group 1 (n = 12 cycles) received no stimulation and women in group 2 (n = 24 cycles) received 150 iu recombinant FSH day(-1) for 3 days, initiated on day 3 after menstruation. Aspiration was performed transvaginally between day 9 and day 17 in the unstimulated group and on day 8 or day 9 in the FSH-primed group after FSH deprivation for 2 or 3 days. All cumulus-enclosed oocytes of healthy appearance were matured in culture medium (TCM-199) in vitro for 28-36 h before intracytoplasmic sperm injection (ICSI). After oocyte retrieval the women were given oestradiol (6 mg day(-1)) and progesterone administration (300 mg day(-1)) was initiated 2 days later. Suitable embryos (maximum two embryos) were transferred on day 3 after ICSI. The percentage of oocytes reaching metaphase II was significantly higher (P < 0.05) in the FSH-primed group (59%, 92/156) compared with the non-primed group (44%, 36/81). There were no significant differences in the rates of oocyte fertilization and cleavage between these groups. No pregnancies were obtained in group 1 (0%, 0/12), whereas seven clinical pregnancies were obtained in group 2 (29%, 7/24) (P < 0.05). In group 2, 37 embryo transfers resulted in eight implantations (21.6%). Three healthy singleton children have been born at term; the remaining pregnancies ended with spontaneous abortions in the first trimester. These results indicate that priming with recombinant FSH before harvesting of immature oocytes from patients with PCOS may improve the maturational potential of the oocytes and the implantation rate of the cleaved embryos.

The impact of the zona pellucida thickness variation of human embryos on pregnancy outcome in relation to suboptimal embryo development. A prospective randomized controlled study.

BACKGROUND: The study represents a prospective randomized controlled study evaluating zona pellucida thickness variation (ZPTV) measurements versus conventional selection of embryos using classic embryo score criteria, prior to embryo transfer in human IVF/intracytoplasmatic sperm injection (ICSI). METHODS: Eighty-six patients having > or =3 embryos, with a classic embryo score of < or =2.2, were allocated to either ZPTV measurement or classic embryo morphology score before embryo transfer. The technician selecting embryos using classic embryo scoring was not aware of the ZPTV measurement results. Of the embryos allocated to ZPTV measurements, only the embryos with the highest ZPTV were transferred. RESULTS: We found no differences in the pregnancy rate per embryo transfer between the two groups (34.4 versus 35.7%). Neither did the implantation rates differ. However, significantly better results were obtained when ZPTV was used as the selection criteria in cases where all embryos had an asynchrony in development or a high embryo score (i.e. were of poorer quality) by classical evaluation (odds ratio = 2.51, confidence interval = 0.33-198). CONCLUSIONS: Using a normally-developed embryo with an optimal embryo score, no beneficial effect of using ZPTV measurement was seen. However, when only less optimal embryos were available to select for transfer, ZPTV provided a x 2.5 increase in the chance of achieving a clinical pregnancy.

Morphology of in-vitro matured oocytes: impact on fertility potential and embryo quality.

BACKGROUND: The purpose of the present study was to investigate the morphology of in-vitro matured metaphase II (MII) oocytes and to observe if there was a difference in the morphology between polycystic and normal ovaries. Furthermore, the morphology of in-vitro matured MII oocytes was related to their subsequent fertilization and cleavage rates and to embryo quality. METHODS: This retrospective study included 264 MII oocytes obtained in 100 consecutive cycles. Oocyte retrieval was performed transvaginally and cumulus enclosed oocytes were matured for 28--30 h before evaluation. Prior to ICSI, all MII oocytes were graded into three groups according to the number of anomalies: grade I: oocytes without any anomaly (n = 144, 54%), grade II: oocytes with one anomaly (n = 87, 33%) and grade III: oocytes with at least two anomalies (n = 33, 12.5%). RESULTS: Oocyte grades did not differ between women with polycystic ovaries [grades I, II and III respectively: 58/94 (61.7%), 29/94 (30.9%) and 7/94 (7.4%)] and women with normal ovaries [grades I, II and III respectively: 86/170 (50.6%); 58/170 (34%); 26/170 (15.3%)]. Morphology was not related to fertilization rates. The cleavage rate was, however, affected by morphological anomalies (grade I [77/144 (53.5%) versus grade II 33/87 (37.9%) (P = 0.03)], although no significant decrease in cleavage rate could be demonstrated when all grade II and III oocytes were compared with normal oocytes. Significantly more embryos of good quality developed after grade I oocytes [54/144 (37.5%)] compared with those from grade II and grade III oocytes (22/120; P = 0.001). The presence of cytoplasmic abnormalities significantly decreased the cleavage rate (P = 0.04) and also the number of good quality embryos (P < 0.001). CONCLUSION: The in-vitro maturation of oocytes without anomalies yields higher quality embryos, with higher cleavage rates, than those with anomalies.

Basal concentrations of oestradiol may predict the outcome of in-vitro maturation in regularly menstruating women.

Retrospectively it was examined whether the number of retrieved oocytes, the maturation rate and cleavage rate can be predicted in regularly menstruating women by the use of the following predictive variables on cycle day 3-4: the concentration of FSH, oestradiol, inhibin B and inhibin A in serum and and the number of ovarian follicles seen by vaginal ultrasound. The study included 132 consecutive aspirations in 100 women attending the clinic for in-vitro maturation due to male factor and/or tubal factor. Fifteen pregnancies were obtained after transfer in 83 cycles, giving a pregnancy rate of 15/132 (11%) per aspiration and 15/83 (18%) per transfer. The concentration of FSH and the number of follicles on day 3 predicted the number of oocytes retrieved, whereas these parameters did not predict the subsequent development of oocytes. No correlation was found between the inhibin B, inhibin A, oestradiol and the number of oocytes respectively. The group with a low concentration of oestradiol on cycle day 3 (threshold <200 pmol/l) (group 1, n = 106 cycles) had a significantly higher pregnancy rate compared to the group with a higher concentration (group 2, n = 26 cycles) (14 versus 0% per aspiration, P = 0.03). The group with a low concentration of oestradiol was subdivided according to the concentration of inhibin A. Group 1a: low inhibin A (threshold <10 pg/ml, n = 84 cycles) and group 1b: high inhibin A concentration (> or =10 pg/ml, n = 19). The pregnancy rate in group 1a (14/84, 17%) differed significantly from group 1b (0/19, 0%) (P = 0.03). It is concluded that a low basal concentration of oestradiol (<200 pmol/l) was shown to be a useful prognostic factor of pregnancy in IVM. The concentration of inhibin A (<10 pg/ml) was of added value.

Embryo quality and developmental potential is compromised by age.

AIMS: The aim of this retrospective study was to assess whether and how the age of the woman affects the quality and developmental potential of the oocytes and embryos in an ART program. METHOD AND MATERIAL: A total of 878 IVF cycles was included as a consecutive series of single transfers (n=292), dual transfers (n=366) and triple transfers (n=220), where all the transferred embryos in each cycle were of identical quality score and identical cleavage stage. RESULTS: We found a highly significant decrease in oocyte recovery with increasing age with about one oocyte per 2.3 years (95% CI 1.8 years to 3.1 years, p<0.0001). Further, we found that the number of oocytes that cleaved declined significantly with increasing age with one per 3.7 years (95% CI 2.7 years to 5.5 years, p<0.0001). This decline was mainly due to the decline in number of oocytes retrieved as the ratio of aspirated oocytes that cleaved with increasing age (approx. -0.04/10 year 95% CI: -0.10; +0.009) was not significantly different (p=0.10). The percentage of transfers using fragmented embryos did not increase significantly with increasing age (p=0.08). The odds of fragmentation increased by 3% per year. The average number of embryos transferred decreased significantly (p=0.03) with age from approximately 2.1 at the age of 25 to approximately 1.8 at the age of 40. In a selected subgroup of embryos all consisting of good quality embryos, a significant decrease was found in implantation rate with increasing age (approx. -0.08/10 years, 95% CI: -1.6; +0.00, p=0.05). Of the 357 pregnancies achieved in this study we found a significantly decreased ongoing pregnancy rate and a significantly increased abortion rate with increasing age (p=0.03). The decrease in the rate of ongoing pregnancies was almost linear, decreasing by approximately 1.5% per year. CONCLUSIONS: We conclude that age has an impact throughout a woman's reproductive life and that it is important to realize that the age-related decline in fertility may start already in the late twenties and not in the mid-thirties as is generally assumed.

Influence of the dominant follicle on in-vitro maturation of human oocytes: a prospective non-randomized study.

The present study was designed to examine the influence of timing of aspiration and the influence of a dominant follicle on maturation and fertility potential of immature oocytes aspirated in unstimulated cycles. The study included 81 regularly cycling women. In group I (n = 53), oocyte retrieval was scheduled the day after a follicle of 10 mm and an endometrium of at least 5 mm were observed. In group II (n = 28), aspiration was scheduled the day after observation of the same ultrasound criteria plus a detected increase (100%) in the level of oestradiol compared with the level on day 3. The maturation rate was significantly higher in group I compared with group II (107/184, 58.2% versus 56/124, 45.2%, P < 0.05), whereas the rates of fertilization and cleavage did not differ between the two groups. The clinical pregnancy rate per aspiration was significantly higher in group I compared with group II (9/53, 17% versus 0/28, 0%, P < 0.05). When comparing oocytes originating from the ovary with the dominant follicle (ipsilateral ovary) with oocytes originating from the ovary without a dominant follicle (contralateral ovary) an increased fertilization rate was observed in group I, and an increased maturation rate was observed in group II. When the data from the two groups were pooled, an increased maturation rate was observed in oocytes originating from the ipsilateral ovary compared with oocytes originating from the contralateral ovary. No difference was found with respect to rates of fertilization and cleavage rates when all oocytes originating from the ipsilateral ovary were compared with all oocytes originating from the contralateral ovary.

Maternal serum supplementation in culture medium benefits maturation of immature human oocytes.

This study compared the rates of maturation, fertilization, cleavage and pregnancy among oocytes matured in medium containing either human serum albumin (HSA) or maternal serum. Immature oocytes were obtained from 51 consecutive regularly cycling women <38 years of age. Immature oocytes were aspirated transvaginally on cycle day 8-9 after priming with FSH (Gonal-F 150 IU/day for 3 days, initiated on day 3). Oocytes were matured in Dyrkningsmedie til IVM supplemented with recombinant FSH (rFSH) 0.075 IU/ml and HCG 0.5 IU/ml for 28-30 h. In group I (n = 63 oocytes obtained from the first 23 cycles) the culture medium was supplemented with 2% (w/v) HSA. In group II (n = 74 oocytes obtained from the following 28 cycles) the medium was supplemented with 10% (v/v) heat-inactivated maternal serum. Intracytoplasmic sperm injection (ICSI) was performed on all methaphase II oocytes. Significantly increased rates of maturation 47/74 (63%) vs. 26/63 (41%) (P < 0.05), pregnancy 6/28 (21%) vs. 0/23 (0%) (P < 0.05) and implantation 6/20 (30%) vs. 0/15 (0%) (P < 0.05) were obtained from oocytes matured in culture medium with maternal serum supplementation compared with oocytes matured in medium supplemented with HSA. These results indicate that factors other than albumin in maternal serum play an important role in maturation and subsequent developmental capacity of human oocytes.

Apoptosis in human cumulus cells in relation to maturation stage and cleavage of the corresponding oocyte.

BACKGROUND: The purpose of this study was to determine the incidence of apoptosis in cumulus cells, and correlate these findings with the maturation stage, fertilization rate and embryo score of the corresponding oocyte, in couples undergoing ICSI due to a male factor. METHODS: The study group consisted of 21 couples where ICSI was performed. The total number of oocyte-cumulus complexes retrieved was 164. Sperm samples were assessed according to the WHO manual, morphology according to the strict criteria and for the presence of apoptosis. The degree of apoptotic DNA fragmentation was determined using the free 3'OH DNA termini in situ with chemically labeled and unlabeled nucleotides. The study was blinded for the technician involved in the assessments of apoptosis in the cumulus cells, apoptosis and morphology in spermatozoa. Ovarian hyperstimulation was carried out according to a long down regulation protocol using GnRH, recFSH and hCG. A maximum of 3 embryos were transferred on day 2 after ICSI. RESULTS: This study demonstrated that the incidence of apoptosis was significantly higher in cumulus cells from germinal vesicle and metaphase I oocytes compared to cumulus cells from metaphase II oocytes (p<0.0001). Non-fertilized metaphase II oocytes showed significantly higher incidence of apoptosis in cumulus cells compared to fertilized metaphase II oocytes (p=0.0082). Furthermore, apoptosis in spermatozoa had an impact on the embryo score (p=0.0087). CONCLUSION: Comparing apoptosis in cumulus cells with maturity of the corresponding oocytes, a significantly higher degree was found related to immature oocytes. Apoptosis in cumulus cells from human metaphase II oocytes impaired the fertilization rate. The degree of fragmentation in the embryo might be correlated to apoptosis in the spermatozoa.

Influence of zona pellucida thickness of human embryos on clinical pregnancy outcome following in vitro fertilization treatment.

PURPOSE: To evaluate the correlation between the degree of zona pellucida thickness variation (ZPTV) of transferred embryos with identical morphologies and subsequent clinical pregnancy rates during 141 intracytoplasmic sperm injection (ICSI) treatment cycles. METHODS: A total of 141 women participating in the study group comprising mostly male factor infertilities and some repeat in vitro fertilization (IVF) failures were transferred, 2 days after ovum pickup, with two to three embryos with identical grades created by fertilization with ICSI. All selected embryos were subjected to zona pellucida thickness measurements immediately prior to transfer using a computerized embryo measurement program from videocinematography recordings. RESULTS: A total of 326 identical-grade transferred pre-embryos resulted in 70 clinical pregnancies with live-born implantation rate of 27.6%. A highly significant correlation was observed between ZPTV of transferred embryos and the IVF outcome with 77.1% and 83.64% of the clinical pregnancies resulting from transferred embryos with ZPTV values greater than 20 and 25, respectively. The mean ZPTV values for 70 conceptual cycles and 62 nonconceptual cycles were 28 +/- 6.43 and 17.85 +/- 8.11, respectively. No significant correlation between ZP thickness and number of blastomeres in the transferred embryos was evident, though embryos with better scores had significantly thinner zonae and higher ZPTV values. Though average zona thickness of embryos declined with age, the mean ZPTV value for women less than 30 years old was significantly higher (25.84 +/- 8.57) as compared with those from women older than 35 years (20.72 +/- 8.45). CONCLUSIONS: The degree of ZPTV of the transferred embryos exhibits a strong correlation with clinical pregnancy outcome following IVF treatment. This potentially reliable indicator of IVF success rate could be used as a criteria for embryo selection during clinical transfers.

The role of DNA strand breaks in human spermatozoa used for IVF and ICSI.

BACKGROUND: The objective of this study was to determine the incidence of spermatozoa with DNA strand breaks in four clinically different groups of infertile couples, and to correlate DNA damage with other semen analysis parameters, as well as fertilization rates and IVF outcome. METHODS: One group consisted of 75 men where the female partners had a tubal obstruction, Group A. Fifty sperm samples were collected from men in unexplained infertile couples, Group B. Fifty men with oligozoospermia and IVF made up Group C. Finally, 61 men with oligozoospermia and where ICSI was performed made up Group D. Sperm samples were assessed according to the WHO manual and for the presence of DNA strand breaks in spermatozoa. The study was blinded for the technician involved in the assessment of DNA strand breaks. IVF was carried out according to a long down regulation protocol using GnRH, FSH and hCG. Embryos were transferred on day 2 after fertilization with a maximum of three embryos. RESULTS: This study demonstrated a negative correlation between the proportion of spermatozoa having DNA strand breaks and the proportion of oocytes fertilized after IVF (p<0.01). Furthermore, the number of spermatozoa with DNA strand breaks was important for the pregnancy rate in the group of unexplained infertile couples. After ICSI no association was found between spermatozoa with DNA strand breaks and fertilization rates (p>0.05). CONCLUSION: DNA strand breaks in human spermatozoa impairs fertilization in both unexplained infertile couples and those with oligozoospermia and IVF. However, after ICSI, this impact of DNA strand breaks were not seen. This creates a specific indication and treatment for this new diagnosed group of otherwise unexplained infertile men.