ICSI outcomes using testicular spermatozoa in non-azoospermic couples with recurrent ICSI failure and no previous live births.

BACKGROUND: The use of testicular over ejaculated spermatozoa for ICSI has been presented as an alternative to overcome infertility in men with poor semen parameters or high levels of sperm DNA fragmentation. OBJECTIVE: To evaluate the efficacy of testicular ICSI outcomes in couples with no previous live birth and recurrent ICSI failure using ejaculated spermatozoa by comparison to the outcomes of couples with similar history of recurrent ICSI using ejaculated spermatozoa only. MATERIALS AND METHODS: A total of 145 couples undergoing ejaculated or testicular ICSI cycles with no previous live births and with at least two previous failed ICSI cycles with ejaculated spermatozoa were evaluated retrospectively. ICSI was performed either with ejaculated (E-ICSI) or with testicular (T-ICSI) spermatozoa. Semen parameters and sperm DNA quality were assessed prior to the oocyte collection day. Primary outcomes included cumulative live birth and pregnancy rates. Secondary analysis included percentage of DNA fragmentation in ejaculated spermatozoa (SCSA® and TUNEL). RESULTS: Patients undergoing T-ICSI (n = 77) had a significantly higher clinical pregnancy rate/fresh embryo transfer (ET) (27.9%; 17/61) and cumulative live birth rate (23.4%; 15/64) compared to patients using E-ICSI (n = 68) (clinical pregnancy rate/fresh ET: 10%; 6/60 and cumulative live birth rate: 11.4%; 7/61). Further, T-ICSI yield significantly better cumulative live birth rates than E-ICSI for men with high TUNEL (≥36%) (T-ICSI: 20%; 3/15 vs. E-ICSI: 0%; 0/7, p < 0.025), high SCSA® (≥25%) scores (T-ICSI: 21.7%; 5/23 vs. E-ICSI: 9.1%; 1/11, p < 0.01), or abnormal semen parameters (T-ICSI: 28%; 7/25 vs. E-ICSI: 6.7%; 1/15, p < 0.01). CONCLUSIONS: The use of testicular spermatozoa for ICSI in non-azoospermic couples with no previous live births, recurrent ICSI failure, and high sperm DNA fragmentation yields significantly better live birth outcomes than a separate cohort of couples with similar history of ICSI failure entering a new ICSI cycle with ejaculated spermatozoa.

Do trained reproductive endocrinologists perform better than their trainees? Comparing clinical pregnancy rates and live birth rates after transfer of single fresh blastocysts.

PURPOSE: To compare clinical pregnancy rates and live birth rates of single blastocyst transfers performed by attending physicians or fellows in reproductive endocrinology and infertility program. METHODS: Retrospective study in an academic reproductive center. We evaluated 932 fresh single blastocyst transfer cycles performed by fellows in training (389 embryo transfers) and by attending physicians (543 embryo transfers). RESULTS: There were no differences in the baseline characteristics and IVF cycle parameters between patients who had transfers performed by fellows or attending physicians. Transfers performed by attending physicians or fellows resulted in similar CPR (46.5 vs. 42.9%, p = 0.28) and LBR (38.3 vs. 34.2%, p = 0.11). Multivariate logistic regression analysis showed that even after adjusting for possible confounders (age, gravity, parity, baseline FSH, antral follicle count, dose of gonadotropins, stimulation protocol, and quality of embryo transferred), CPR (OR 0.81, CI 0.62-1.07) and LBR (OR 0.79, CI 0.6-1.05) in the two groups were comparable. CONCLUSION: Clinical pregnancy rate and live birth rate after embryo transfer performed by attending staffs or fellows are comparable. This finding reassures fellowship programs that allowing fellows to perform embryo transfers does not compromise the outcome.

Does using testicular sperm retrieval rather than ejaculated spermatozoa improve reproductive outcomes in couples with previous ART failure and poor ovarian response? A case-controlled study.

The objective of this study was to assess whether testicular-retrieved spermatozoa improve reproductive outcomes compared to fresh ejaculate in women with poor ovarian response and a history of previous ART failure. The study was performed as a retrospective case-control study at a university-based reproductive center in Montreal, Quebec, Canada. Eighteen poor-responder patients were matched 3 : 1 with 54 controls. Poor responders were defined as those with ≤3 oocytes retrieved at oocyte pickup. Cases were identified as poor responders, and only those with previous IVF failure(s) as an indication for testicular-retrieved spermatozoa were included. Controls were age and cycle attempt number matched. All patients were included only once. From January 1, 2009 to December 31, 2015, all patients and controls underwent an IVF cycle using ICSI with either testicular spermatozoa or ejaculated spermatozoa, respectively. Outcomes included live birth rate, pregnancy rate, miscarriage rate, oocyte number, and embryo transfer (ET) day. The results showed live birth rates, pregnancy rates, and miscarriage rates were similar. There were fewer day 2 ETs (8.5% vs. 48.6%, p = 0.01) and more day 5 blastocyst transfers (25.0% vs. 5.4%, p = 0.05) in the testicular sperm retrieval group compared to controls and thus an overall suggestion of better embryo quality in the testicular sperm group. Overall, however, the use of testicular sperm retrieval appears to add little. Women with poor ovarian response typically have a poor prognosis with respect to live birth rates, and this is further supported in this study. The suggestion of better embryo quality in the testicular-retrieved sperm group would need to be further assessed in a larger multicentered study.

The relationship between fat and progesterone, estradiol, and chorionic gonadotropin levels in Quebec cow's milk.

PURPOSE: The majority of milk in industrialized countries is obtained from pregnant cows, which contains increased levels of estrogen and progesterone compared to non-pregnant cows. The aim of this study was to quantify the amount of hormones present in milk with different fat content because previous studies on humans have shown potential effects of increased milk consumption on serum and urine hormone levels as well as on sperm parameters. However, it is unclear whether consumption of milk at the currently recommended levels would lead to systemic effects. METHODS: Samples of cow's milk of varying fat concentrations (0, 1, 2, 3.25, 10, and 35%) were analyzed via competitive ELISA assays. RESULTS: Progesterone concentrations were significantly correlated to increasing fat content of milk (r = 0.8251, p = 0.04). CONCLUSIONS: Research on conditions in which additional progesterone may have an effect on human health should consider inclusion of limitation of milk intake and its effects. Further studies are needed to determine the concentration of progesterone in milk of different fat content in other regions and countries and to quantify the potential pathophysiologic role.

Obstetric outcomes of IVF trichorionic triamniotic triplets which are spontaneously or electively reduced to twins.

PURPOSE: To assess the obstetric outcome of IVF triplets which are spontaneously or electively reduced to twins. METHODS: Retrospective study of trichorionic triplets (TT) and dichorionic twins (DT) conceived with IVF. RESULTS: Compared to TT without reduction, TT with spontaneous reduction (SR) (OR: 5.6, 95% CI: 1.6-19.9) or elective reduction (ER) (OR: 14.0, 95% CI: 3.92-50.02) to twins were significantly more likely to be delivered at ≥34 weeks. Compared to DT likelihood of delivery ≥34 weeks was similar for SR and ER. Compared to TT without reduction, the risk of pregnancy loss before 24 weeks was not increased with SR or ER. Compared to DT the risk of pregnancy loss before 24 weeks was not significantly increased for SR or ER. CONCLUSION: Obstetric outcome of IVF triplets with SR is better than ongoing triplets and is similar to that of ER of TT and DT.

Mechanisms of follicle selection and development.

Follicle recruitment and selection, the process that gives rise to the dominant follicle (DF) and the physiological state of the DF are important areas of research. The selection of a single ovarian follicle for further differentiation and finally ovulation is a shared phenomenon in monovulatory species including humans. The DF is different from other follicles because it can escape atresia (the fate of all other follicles). The DF cells develop corpus luteum if exposed to the luteinizing hormone (LH) surge. Several mechanisms for DF selection have been proposed. Rising follicle stimulating hormone (FSH) concentrations induce the emergence of a follicle wave and cohort attrition occurs during declining FSH concentrations, resulting in DF selection. Cohort secretions are initially responsible for the decline in FSH, which is subsequently suppressed by the selected DF lowering it below the threshold of FSH requirements of all other cohort follicles. The DF acquires relative FSH-independence in order to continue growth and differentiation during further declining FSH concentrations. A transition from FSH- to LH-dependence is postulated as the mechanism for the continued development of the selected DF. In addition, FSH and insulin-like-growth factor (IGF) enhance each other's ability to stimulate follicle cell function. Access of IGF II to their receptors is regulated by IGF binding proteins that are in turn regulated by specific proteases; all of which have been ascribed a role in DF development. One other shared mechanism recently proposed for DF selection is the possible differential regulation of blood vessel formation. Anti-Müllerian hormone (AMH) also plays a critical role in selection of the DF. AMH levels decline as the size of the follicle increases. Once follicles reach a size at which they are dominant, it has largely disappeared. From the time a follicle has been selected, the follicle destined to ovulate greatly enlarges and shows marked changes in its steroidogenic activity. LH surge causes a significant decline in gap junctions leading to dissociation of mural granulosa cells (GC) and expansion of the cumulus-oocyte complex (COC). The oocyte resumes its meiosis and progresses from prophase 1 to metaphase 2 at the time of ovulation. The concept of DF selection could be applied to the in-vitro maturation (IVM) program. Understanding the mechanism of DF selection in menstrual cycles is the key to planning the optimal timing of oocyte retrieval in order to obtain competent oocytes and embryos. Although the timing of oocyte retrieval is still open to debate, there is evidence to suggest that it may be better to retrieve oocytes before the small cohort follicles complete the process of atresia following selection of the DF.

A unique biological in-vivo model to evaluate follicular development during in-vitro maturation treatment.

The aim of this study was to identify the size in which the dominant follicle acquires the ability to produce a functional corpus luteum. This observational study includes 15 women with ovulatory cycles who underwent human chorionic gonadotrophin (HCG)-primed in-vitro maturation (IVM) treatments without embryo transfer. All patients received subcutaneous injection of HCG 10,000 IU 38 h prior to oocyte retrieval. Five to seven days following retrieval, serum concentrations of progesterone and oestradiol were measured along with ultrasound scan measuring the antral follicle count. Using receiver operating characteristic curves and the Youden index (J), this study clearly shows that the diameter of the dominant follicle at the time of the LH surge is a good predictor for its ability to form a progesterone-producing corpus luteum (area under the curve 0.94). These findings revealed that the dominant follicle develops the competence to form a corpus luteum, signified by the production of more than 10 nmol/l serum progesterone at 5-7 days from oocyte retrieval, as soon as it acquires a diameter of 10.5-12.0mm. In addition, a new cohort of viable antral follicles can be identified as early as 5-7 days following IVM oocyte retrieval.

Fertility preservation treatment for young women with autoimmune diseases facing treatment with gonadotoxic agents.

OBJECTIVE: To describe a case series of seven women with SLE and other systemic autoimmune rheumatic diseases (SARDs) who required cyclophosphamide therapy and underwent fertility preservation treatments. METHODS: Of the seven patients reported here, five women had SLE with nephritis, the sixth had immune thrombocytopenia purpura (ITP) and the seventh had microscopic polyangiitis (MPA) with renal involvement. All women were nulliparous and younger than 35 yrs. RESULTS: Patients with SLE underwent in vitro maturation (IVM) of immature oocytes aspirated during a natural menstrual cycle followed by vitrification of the matured oocytes if a male partner was not available, or vitrification of embryos if one was available. The patient with ITP and the patient with MPA underwent gonadotropin ovarian stimulation followed by oocyte or embryo vitrification. All women completed fertility preservation treatment successfully and mature oocytes or embryos (36 and 13, respectively) were vitrified. No complications were associated with this treatment and cytotoxic therapy was initiated as scheduled in all cases. CONCLUSIONS: Oocyte or embryo cryopreservation should be considered for fertility preservation in young women with SARDs who face imminent gonadotoxic treatment. In patients, where gonadotropin ovarian stimulation is deemed unsafe, IVM of immature oocytes, aspirated during a natural menstrual cycle, followed by vitrification or fertilization of the mature oocytes, seems to be safe and feasible. For patients in whom hormonal ovarian stimulation is not contraindicated, this method may be considered depending on the urgency to start cytotoxic therapy.

Pregnancies resulting from in vitro matured oocytes collected from women with regular menstrual cycle.

PURPOSE: To demonstrate that human immature oocytes retrieved from women with regular menstrual cycles can undergo maturation and fertilization, and that the resulting embryos can establish pregnancies. METHODS: Immature oocytes (n = 568) were retrieved from women with regular menstrual cycle. The intact immature oocytes (n = 506) were allowed to mature in YS medium supplemented with 70% human follicular fluid (hFF); the matured oocytes were fertilized with husband sperm. Two pronuclei oocytes were cocultured with cumulus cells in YS medium supplemented with 10% hFF until 2 or 3 days after insemination. The cleaved embryos were transferred in uteri. RESULTS: Follicles were aspirated on Day 9.2 +/- 5.3 of 63 natural cycles from 51 patients (mean age = 34.8 +/- 4.0 years). The average number of retrieved immature oocytes was 9.0. The maturation rate was 74.3% (376/506). The two PN and cleavage rates were 72.6% (273/376) and 89.0% (243/273), respectively. Embryo transfer was achieved in 51 cycles and clinical pregnancy rate was 17.6% (9/51). CONCLUSIONS: The results suggest that in vitro matured oocytes can undergo fertilization and the resulting embryos may successfully lead to pregnancies. However, further research is needed to improve IVM technique to achieve success rate comparable to gonadotrophin stimulated cycles.

Alternative embryo transfer on day 3 or day 5 for reducing the risk of multiple gestations.

PURPOSE: This study was carried out to reduce the possibility of high-order multiple gestations and the failure of embryo transfer by determining their replacement date based on the number and quality of 2-day embryos. METHODS: All zygotes were cocultured with cumulus cells in 10 microliters of YS medium containing 10% human follicular fluid (hFF) for 48 or 96 hr. In period I, all embryos were transferred on day 3 (1032 cycles). In period II, the embryos were transferred on either day 3 or day 5 by determining their replacement date based on the number and quality of 2-day embryos: there were 2701 patients in whom embryos were replaced on day 3 (in the case that the number of zygotes was less than eight and the number of good-quality embryos was less than three) and 1952 patients less than 40 years old in whom embryos were replaced on day 5 (in the case that the number of zygotes was eight or more and/or the number of good-quality embryos was three or more). On the other hand, patients who were 40 years old or more were alloted to day 3 transfer cycles, regardless of the number and quality of the 2-day embryos, due to the possibility of their not producing blastocyst-stage embryos in vitro. RESULTS: The number of embryos transferred in period II was 2.9 +/- 0.6, while that in period I was 3.7 +/- 0.5. The multiple pregnancy rate was significantly decreased in period II (30.7%) compared to that (49.6%) in period I, while the pregnancy and implantation rates in period II (36.1 and 16.4%, respectively) were not lower than those (34.9 and 16.1%, respectively) in period I. The rate of triplet or more gestations was significantly minimized in period II (2.3%) compared to that in period I (26.5%). CONCLUSIONS: We propose that determination of the date on which embryos should be transferred based on the number and quality of embryos on day 2 may help to maintain an acceptable pregnancy rate, while minimizing embryo transfer failure and high-order multiple gestations.

Acrosome reaction of human spermatozoa is mainly mediated by alpha1H T-type calcium channels.

The objectives of this study were: (i) to investigate the possible role of T-type Ca(2+) channels on the acrosome reaction (AR) of human spermatozoa; and (ii) to determine the sub-type of T-type calcium channels involved in the AR. The AR was induced in vitro by mannose-bovine serum albumin (BSA). The inhibitory effects of mibefradil (T-type Ca(2+) channel blocker), NiCl(2), or nifedipine (L-type Ca(2+) channel blocker) on the mannose-BSA induced AR were evaluated in capacitated human spermatozoa. The AR was sensitively inhibited by low micromolar concentrations of mibefradil (IC(50) = 1 micromol/l) in a dose-dependent manner. Low concentrations of Ni(2+) (IC(50) = 40 micromol/l) also inhibited the mannose-BSA induced AR. On the contrary, higher concentrations of nifedipine were required to block AR (IC(50) = 60 micromol/l). Reverse transcription-polymerase chain reaction (RT-PCR) was performed to identify the sub-types of T-type channels present in human testes. Analysis of PCR products showed that only alpha1H subunits are expressed in testes. The expression of the alpha1H subunit may be tissue specific since its mRNA was not detected in the human ovary. The present study suggests that the AR of human spermatozoa is highly associated with T-type Ca(2+) channels and is mainly mediated by calcium influx through alpha1H T-type Ca(2+) channels.

Repression of hspA2 messenger RNA in human testes with abnormal spermatogenesis.

OBJECTIVE: To evaluate the messenger RNA (mRNA) expression of hspA2 in testes of infertile men with azoospermia. DESIGN: Prospective study. SETTING: Center for Reproduction and Genetics, Pundang Je-Saeng General Hospital, Dae-Jin Medical Center, Korea. PATIENT(S): Azoospermic patients (n = 15) undergoing testicular biopsy for pathologic evaluation were selected. INTERVENTION(S): After pathologic evaluation, testicular biopsy specimens were subdivided into three groups: group 1, normal spermatogenesis (n = 5); group 2, spermatocyte arrest (n = 5); and group 3, Sertoli cell-only syndrome (n = 5). The levels of hspA2 mRNA expression were compared in testes of group 1, group 2, and group 3 with the use of a competitive reverse transcription polymerase chain reaction (RT-PCR) technique. MAIN OUTCOME MEASURE(S): Comparison of hspA2 mRNA levels in testes. RESULT(S): On competitive RT-PCR analyses for hspA2 mRNA, significant hspA2 expression was observed in group 1, whereas a very low level of hspA2 was expressed in groups 2 and 3. CONCLUSION(S): This study demonstrates that hspA2 gene expression is down-regulated in human testes with abnormal spermatogenesis, which in turn suggests that the hspA2 gene might play a specific role during meiosis in human testes.

Specific expression of heat shock protein HspA2 in human male germ cells.

In the mouse, the heat shock protein 70-2 (Hsp70-2) has been found to play a critical role in spermatogenesis. The HspA2 gene is the human homologue of the murine Hsp70-2 gene with 91.7% identity in the nucleotide coding sequence. We examined the expression of HspA2 in human tissues. To detect HspA2 expression, antiserum 2A that was raised against mouse Hsp70-2 and that cross-reacted with human HspA2 protein expressed in Escherichia coli was used. The results of Western blotting indicate that significant HspA2 expression occurs in testes with normal spermatogenesis, whereas only a low amount of HspA2 was expressed in testis with Sertoli cell-only syndrome. Only a small amount of HspA2 was detected in breast, stomach, prostate, colon, liver, ovary, and epididymis. Immunoreactivity to HspA2 was present in spermatocytes and spermatids in the testes with normal spermatogenesis, while immunoreactivity to HspA2 in testis with Sertoli cell-only syndrome was remarkably decreased or inconspicuous over the entire cell. These results demonstrate that the HspA2 protein is highly expressed in human male specific germ cells, suggesting that HspA2 protein may play a specific role during meiosis in human testes as found in the murine model.

Chromosome and spindle configurations of human oocytes matured in vitro after cryopreservation at the germinal vesicle stage.

OBJECTIVE: To investigate effects of cryoprotectant and cryopreservation on the chromosome and microtubule configuration of human immature oocytes. DESIGN: Intact cumulus-enclosed immature oocytes were collected from unstimulated ovaries and divided into three groups: group 1, no treatment (control); group 2, only 1,2-propanediol treatment, and group 3, cryopreserved oocytes. Oocytes in groups 1 and 2, and oocytes that survived after cryopreservation in group 3 were cultured for 48 hours. SETTING: Infertility Medical Center at the CHA General Hospital, Seoul, Korea. PATIENT(S): Oocytes were obtained from patients undergoing gynecologic surgery. MAIN OUTCOME MEASURE(S): Maturation rate and abnormality in chromosomes by fluorescence in situ hybridization and in the spindle by immunostaining for tubulin. RESULT(S): There was no effect of propanediol-only treatment on the chromosomal (41.4%) and spindle abnormalities (35.3%) in group 2 compared with control oocytes (31.8% and 22.2%, respectively), whereas a statistically significant increase in abnormalities in chromosomes (77.8%) and spindles (70%) was found in group 3. CONCLUSION(S): Human oocytes matured in vitro after cryopreservation at the germinal vesicle stage showed increased incidence of chromosomal and spindle abnormalities. These abnormalities may impair the capacity for further development of the embryos derived from frozen-thawed oocytes.

Effects of 1,2-propanediol and freezing-thawing on the in vitro developmental capacity of human immature oocytes.

OBJECTIVE: To investigate effects of 1,2-propanediol and freezing-thawing treatment on the maturation and developmental capacity of the human immature oocytes obtained from unstimulated ovaries. DESIGN: Intact cumulus-enclosed immature oocytes collected from unstimulated ovaries were divided into three groups, such as no treatment as control (group 1), only 1,2-propanediol-treated (group 2), and cryopreserved group (group 3). Oocytes in group 1, group 2, and survived oocytes from cryopreservation in group 3 were cultured for 48 hours. A random selection of matured oocytes was inseminated with normal donor sperm to evaluate the fertilization and developmental capacity. SETTING: Infertility Medical Center at the CHA General Hospital, Seoul, Korea. PATIENT(S): Oocytes were obtained from patients undergoing gynecological surgery. MAIN OUTCOME MEASURE(S): Rates of survival, maturation to metaphase II, fertilization, and cleavage. RESULT(S): Survival rate after freezing-thawing in group 3 was 55.1% (54/98). Oocytes were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 20% fetal bovine serum, 10 IU/mL pregnant mare serum gonadotropin, and 10 IU/mL hCG. Maturation rates were 76.8% (63/82), 67.1% (47/70), and 59.3% (32/54) in the groups 1, 2, and 3, respectively. Maturation rate in group 3 was significantly lower than that of group 1. Fertilization rates were 90.5% (19/21), 81.0% (17/21), and 42.9% (6/14), and cleavage rates were 94.7% (18/19), 88.2% (15/17), and 16.7% (1/6) in groups 1, 2, and 3, respectively. Fertilization and cleavage rates of survived oocytes in group 3 also were significantly lower than those of groups 1 and 2. CONCLUSION(S): Results suggest that the pretreatment with 1.5 M 1,2-propanediol itself before the freezing has no inhibitory effect on the maturation, fertilization, and cleavage of human immature oocytes in vitro. However, the freezing-thawing procedure used had detrimental effects on the maturation and developmental capacity.

Intestinal trematode infections in the villagers in Koje-myon, Kochang-gun, Kyongsangnam-do, Korea.

An epidemiological study for the intestinal trematode infection of the villagers was done in Koje-myon, Kochang-gun, Kyongsangnam-do, Korea in March, 1994. Of 116 stool specimens examined, total helminthic ova positive cases were 13 (11.2%) and cumulative ova positive cases 21 (18.1%): Echinostoma hortense 11 cases (9.5%), Metagonimus sp. 6 cases (5.2%), and Clonorchis sinensis 4 cases (3.4%). After the treatment and purgation, variable numbers of E. hortense, 6 to 227 per person, were collected from 7 echinostome egg positive cases, together with M. takahashii in 6 cases. Three adult flukes of E. cinetorchis were collected from one person and one Stellantchasmus falcatus was recovered from another case. This mountainous village was proved to be one of the endemic foci of echinostomiasis in Korea.