The exact synchronization timing between the cleavage embryo stage and duration of progesterone therapy-improved pregnancy rates in frozen embryo transfer cycles: A cross-sectional study

Background: Synchronization between the embryonic stage and the uterine endometrial lining is important in the outcomes of the vitrified-warmed embryo transfer (ET) cycles. Objective: The aim was to investigate the effect of the exact synchronization between the cleavage stage of embryos and the duration of progesterone administration on the improvement of clinical outcomes in frozen embryo transfer (FET) cycles. Materials and Methods: 312 FET cycles were categorized into two groups: (A) day- 3 ET after three days of progesterone administration (n = 177) and (B) day-2 or -4 ET after three days of progesterone administration (n = 135). Group B was further divided into two subgroups: B1: day-2 ET cycles, that the stage of embryos were less than the administrated progesterone and B2: day-4 ET cycles, that the stage of embryos were more than the administrated progesterone. The clinical outcome measures were compared between the groups. Results: The pregnancy outcomes between groups A and B showed a significant differences in the chemical (40.1% vs 27.4%; p = 0.010) and clinical pregnancies (32.8% vs 22.2%; p = 0.040), respectively. The rate of miscarriage tended to be higher and live birth rate tended to be lower in group B than in group A. Also, significantly higher rates were noted in chemical pregnancy, clinical pregnancy, and live birth in group A when compared with subgroup B2. Conclusion: Higher rates of pregnancy and live birth were achieved in day-3 ET after three days of progesterone administration in FET cycles. Key words: Endometrium, Embryo transfer, Pregnancy, Live birth, Progesterone

Pentoxifylline treatment as a safe method for selecting viable testicular spermatozoa before cryopreservation of a small numbers of spermatozoa in azoospermia individuals

Background: Single sperm cryopreservation (SSC) is a specific technique especially used in individuals with small numbers of sperm who suffered from non-obstructive azoospermia (NOA). Testicular specimens possess poor motility and low population of viable spermatozoa. Therefore, sperm selection methods such as applying pentoxifylline (PTX) may improve motility in these cases. The main aim of this study was to evaluate the protective effects of PTX on testicular spermatozoa before and after performing SSC. Methods: Thirty testicular samples were obtained from men with azoospermia. This study was conducted in two phases. Phase 1 evaluated the effect of PTX for sperm selection before SSC. Twenty testicular samples were divided to two experimental groups: SSC without (I) and with PTX treatment (II). For PTX treatment spermatozoa were incubated with PTX at 37°C for 30 min and only motile spermatozoa were selected for SSC. In phase 2, ten testicular samples were cryopreserved with SSC and warming procedure was carried out in droplet with and without PTX. Motility and viability rates, morphology by motile sperm organelle morphology examination (MSOME), DNA fragmentation by sperm chromatin dispersion test (SCD) and mitochondrial membrane potential (MMP) were evaluated. Results: In phase 1, post warm motility rate was higher in PTX exposed group compared to the unexposed group (25.6 ± 8.13 vs. 0.85 ± 2.1) (p > 0.00). Recovery rate, viability and morphology were not significantly different between groups. DNA integrity and MMP were also similar between both groups. In phase 2 although motility increased in PTX group compared to without PTX group (29.30 ± 12.73 vs. 1.90 ± 2.64) (p > 0.00), the viability rate was not different (70.40 ± 12.12 vs. 65.30 ± 11.87). All above mentioned parameters were similar between the two SSC groups. Conclusions: Supplementation of testicular spermatozoa with PTX before cryopreservation increases motility and did not have adverse effects on viability, morphology, DNA integrity and MMP. PTX could be used as sperm selection method before single sperm cryopreservation, but PTX could not maintain motile the most of viable testicular sperms

Biological and physiological characteristics of human cumulus cell in adherent culture condition

Background: Cumulus cells, as oocyte nurse cells, provide a suitable microenvironment with growth factors and cellular interactions required for oocyte maturation. Thus, these cells may serve as a natural niche for in vitro studies of female germ cell development. Cumulus cells may help attain a better understanding of the causes of infertility in women and eventually improve the outcomes of cases that respond poorly to standard infertility treatment. Objective: The aim of this study was to isolate, culture, and investigate the biological characteristics of human cumulus cells. Materials and Methods: In this experimental study, cumulus cells were isolated, cultured, and characterized using reverse transcription-polymerase chain reaction analyses of specific genes including FOXL2, CYP19A1, FSHR, AMHR, and LHR. The presence of vimentin, a structural protein, was examined via immunofluorescent staining. Moreover, levels of anti-mullerian hormone (AMH) and progesterone secretion by cumulus cells were measured with ELISA after 2, 4, 12, 24, and 48 hr of culture. Results: In adherent culture, human cumulus cells expressed specific genes and markers as well as secreted AMH and progesterone into the medium. Conclusion: Cumulus cells secrete AMH and progesterone in an adherent culture and might be applicable for in vitro maturation (IVM) and in vitro gametogenesis (IVG) studies. Key words: Cumulus cells, Conditioned medium, In vitro maturation, In vitro gametogenesis, Niche

Vitrification Increased Vacuolization of Human Spermatozoa: Application of MSOME Technology

Abstract Background: Sperm vitrification is a technique of ice and cryoprotectant free cryopreservation by direct plunging of sperm suspension into liquid nitrogen (LN2). The aim of this study was to investigate the influence of cryoprotectant free-vitrification on human sperm fine structure by MSOME technology and the fertility potential by zona binding assay (ZBA). Methods: 20 normo-ejaculates were prepared by swim up technique, and supernatants were divided into two parts of fresh and vitrified groups. For vitrification, sperm was dropped into LN2. Sperm motility, morphology, viability and MSOME were evaluated for each sample. In MSOM morphologically normal sperm (class 1), ≤2 small vacuoles (class 2), and one large vacuole or >2 small vacuoles (class 3) were evaluated. Also, fertility potential was evaluated by zona binding assay. Data was analyzed using paired t-test or Willcoxon's test and p-value <0.05 was considered significant. Results: Vitrification significantly reduced both progressive motility, viability and morphology. Also, normal morphology of spermatozoa decreased significantly after vitrification. In MSOME evaluation, normal motile spermatozoa (Class 1) decreased from 23.00±12.44 to 16.00.56±10.79 after vitrification (p=0.008). Although spermatozoa classes 2 and 3 were increased, the difference was not significant. Moreover, fertility potential of motile spermatozoa was reduced after vitrification (9.0±13.87 vs. 13.40±22.73; p=0.07). Conclusion: Vitrification increased the rate of vacuolization in motile sperm head. Therefore, MSOME technology is recommended for assessment of sperm fine morphology in ICSI program used cryopreserved spermatozoa

Morphology and viability of human spermatozoa vitrified with a new, cryoprotectant-free, artificial seminal fluid

Cryopreservation is a process finalized to store tissues and cells at a very low temperature. The most common freezing protocols used for gamete preservation in Assisted Reproductive Technologies are slow freezing and vitrification (1). Vitrification combines ultrarapid cooling with high concentrations of cryoprotectants; it avoids, better than slow freezing, the formation of ice crystals. It has been demonstrated, however, that cryoprotectant addition may significantly reduce cell viability (2). This study was aimed to design a new, cryoprotectant-free, medium similar to normal human seminal fluid (SF) formulation (artificial seminal fluid; ASF), and to compare the cryoprotective potential of this medium with SF and Human tubal fluid (HTF) medium. Thirty normal ejaculates were processed with swim-up technique and sperm suspensions were divided in four groups: fresh (controls); vitrified in HTF (Vit HTF); vitrified in patients’ SF (Vit SF); and vitrified in ASF (Vit ASF). To identify the effects of the different media we assessed sperm parameters of motility, viability and morphology after warming. Spermatozoa ultrastructure was also evaluated by scanning and transmission electron microscopy (SEM and TEM). The results showed that sperm motility, viability and normal morphology were significantly higher in Vit ASF than in Vit HTF. The same parameters were better in Vit ASF than in Vit SF, but only viability differed significantly. Deep cytoplasmic invaginations and folded tails were commonly observed by SEM in all vitrified sperms, but this alterations were more evident in Vit HTF and Vit SF than in Vit ASF. By TEM, acrosome damage, plasma membrane loss, chromatin vacuolation, disruption of mitochondria and adherence of several tail sections together were observed in all vitrified groups; the latter phenomenon, however, was more evident in Vit HTF and Vit SF than in Vit ASF. In conclusion, vitrification of human spermatozoa with ASF seems more effective in preserving sperm quality than Vit SF and, particularly, Vit HTF

Revealing the secret life of pre-implantation embryos by time-lapse monitoring: a review

High implantation success following in vitro fertilization cycles are achieved via the transfer of embryos with the highest developmental competence. Multiple pregnancies as a result of the transfer of several embryos per cycle accompany with various complication. Thus, single-embryo transfer (SET) is the preferred practice in assisted reproductive technique (ART) treatment. In order to improve the pregnancy rate for SET, embryologists need reliable biomarkers to aid their selection of embryos with the highest developmental potential. Time-lapse technology is a noninvasive alternative conventional microscopic assessment. It provides uninterrupted and continues the survey of embryo development to transfer day. Today, there are four time-lapse systems that are commercially available for ART centers. In world and Iran, the first time lapse babies were born in 2010 and 2015, respectively, conceived by SET. Here, we review the use of time-lapse monitoring in the observation of embryogenesis as well as its role in SET. Although, the findings from our review support common use of time-lapse monitoring in ART centers; but, future large studies assessing this system in well-designed trials are necessary

Missed estradiol determination resulting in oocyte retrieval and embryo development following controlled ovarian hyperstimulation at early pregnancy: Case report

This paper is a case report on the success of oocyte retrieval and good quality embryo development following controlled ovarian hyperstimulation at early pregnancy. A 30-year-old patient underwent controlled ovarian hyperstimulation by gonadotropin-releasing hormone agonist long protocol. On the day of oocyte collection, a 5-week gestational sac was observed by exact sonography monitoring. However, via ultrasound guided follicle puncture, 7 oocytes were collected. After intarcytoplasmic sperm injection, 3 developed good quality embryos were cryopreserved. Moreover, the natural pregnancy was continued and finally a healthy live birth was achieved. Despite physiological hormonal changes during pregnancy, the follicular growth occurred and followed by oocyte retrieval and embryo development, subsequently

Quality of testicular spermatozoa improves with changes in composition of culture medium

Abstract Background Spermatozoa retrieved from the testis and epididymis are deprived of the beneficial effects of seminal fluid. Thus applying an artificial medium with normal seminal fluid characteristics, known as artificial seminal fluid (ASF), may provide an appropriate condition for improving some sperm parameters in azoospermia. The objective was to investigate the impact of in vitro exposure of testicular and epididymal spermatozoa to ASF on sperm quality. The study was conducted on testicular (n = 20) and epididymal (n = 20) sperm specimens obtained from azoospermic men. Each sample was divided into two equal parts: Part I) for processing and incubation with Ham’s F10 medium; Part II) for processing and incubation with ASF. Results After 2 h incubation, testicular sperm motility was significantly higher in ASF than in Ham’s F10 medium. In comparison to 0 h, mitochondrial membrane potential levels of testicular spermatozoa were significantly higher after 2 h and 24 h in ASF and after 24 h in Ham’s F10 medium. Furthermore, the data indicated significantly lower rates of epididymal spermatozoa with high MMP in both media after 24 h. There were no significant differences in the DNA fragmentation index of testicular and epididymal spermatozoa between ASF and Ham’s F10 medium at different time points. Conclusion The results demonstrated that in vitro incubation of testicular spermatozoa improved their motility more effectively than Ham’s F10 medium in the short term (2 h), but had no effect on epididymal spermatozoa. Since the physiology of testicular spermatozoa is different from that of ejaculated spermatozoa, it seems that a special environment should be designed and used for each of them

Embryo cryopreservation following In-vitro maturation for fertility preservation in a woman with Mullerian adenosarcoma: A case report

In-vitro maturation (IVM) of the immature oocytes recovered from the surgically removed ovarian tissue has been considered as a process for fertility preservation in patients with cancer. Fertility preservation for a woman with Mullerian adenocarcinoma. A 37-year-old woman with Mullerian adenocarcinoma was a candidate for ovarian resection. The immature oocytes were retrieved after ovarian resection from a 37-year-old woman with Mullerian adenocarcinoma. The oocytes underwent IVM and were fertilized using intracytoplasmic sperm injection (ICSI). Two healthy embryos were cryopreserved for future use. The immature oocytes from the ovarian tissue can be matured with IVM for generation of embryos after ICSI. The embryos can be vitrified using routine methods for fertility preservation in young women with cancer

The Exact Synchronization Timing Between the Cleavage Embryo Stage and Duration of Progesterone Therapy-improved Pregnancy Rates in Frozen Embryo Transfer Cycles: A Cross-sectional Study

Background: Synchronization between the embryonic stage and the uterine endometrial lining is important in the outcomes of the vitrified-warmed embryo transfer (ET) cycles. Objective: The aim was to investigate the effect of the exact synchronization between the cleavage stage of embryos and the duration of progesterone administration on the improvement of clinical outcomes in frozen embryo transfer (FET) cycles. Materials and Methods: 312 FET cycles were categorized into two groups: (A) day- 3 ET after three days of progesterone administration (n = 177) and (B) day-2 or -4 ET after three days of progesterone administration (n = 135). Group B was further divided into two subgroups: B1: day-2 ET cycles, that the stage of embryos were less than the administrated progesterone and B2: day-4 ET cycles, that the stage of embryos were more than the administrated progesterone. The clinical outcome measures were compared between the groups. Results: The pregnancy outcomes between groups A and B showed a significant differences in the chemical (40.1% vs 27.4%; p = 0.010) and clinical pregnancies (32.8% vs 22.2%; p = 0.040), respectively. The rate of miscarriage tended to be higher and live birth rate tended to be lower in group B than in group A. Also, significantly higher rates were noted in chemical pregnancy, clinical pregnancy, and live birth in group A when compared with subgroup B2. Conclusion: Higher rates of pregnancy and live birth were achieved in day-3 ET after three days of progesterone administration in FET cycles. Key words: Endometrium, Embryo transfer, Pregnancy, Live birth, Progesterone