The spermatoozoon is an excitable cell that responds to specific effectors by rapidly changing its behaviour. In species with external fertilisation, spermatozoa are stored in a quiescent state in the testis, but within seconds after spawning, dilution into water triggers several activation events such as increases in motility and respiration. In some species, these are followed by the acrosome reaction, an exocytotic process that allows the spermatozoon to penetrate the egg investments and activate the egg (Dale, 1983). The majority of information on sperm activation has come from the sea urchin; secondarily teleosts and starfish have proved to be useful models.

The oocyte is known from recent studies in the mouse, cow, sheep and human to be a central regulator of follicular cell function. However, in the pig, little information is known about the regulation of cumulus expansion by oocyte-secreted factors and oocyte quality. We investigated the possible effects of oocyte-secreted factors during in vitro maturation on cumulus expansion and on porcine oocytes as judged by subsequent embryonic development after parthenogenetic activation. Cumulus–oocyte complexes (COC) from antral follicles of pig ovaries collected from a local abattoir were divided into control and treatment groups and were cultured in tissue culture medium 199 supplemented with follicle-stimulating hormone. Treatment groups consisted of increasing numbers of denuded oocytes (DO) co-cultured with COC (at ratios of COC to DO of 1:1, 1:2, 1:3, 1:4 and 1:5). After incubation for 44 h, cumulus expansion and maturation rates were assessed and oocytes were activated parthenogenetically. Cumulus expansion in the 1 COC:4 DO and 1 COC:5 DO groups was low and altered because full dispersion of the outer layer did not occur. Cell viability was not affected, as measured by the automated cell counter, but scanning electron microscopy revealed only a scanty extracellular matrix. Blastocyst rate was significantly higher in the 1 COC:4 DO (34.4%) and in the 1 COC:5 DO (34.9%) groups (p < 0.05) when compared with other groups. Maturation rate, cleavage rate and total cell number showed no significant difference between control and treatment groups. Amplification by reverse transcription polymerase chain reaction (RT-PCR) showed up-regulation of growth differentiation factor 9 (GDF9) in the cumulus cells in the 1 COC:4 DO group at 44 h. We conclude that denuded porcine oocytes could improve the maturation of COC as evidenced by increased blastocyst development in the 1 COC:4 DO, even though cumulus expansion was poor. This improvement could be a result of the GDF9 up-regulation.

In vitro culture of the embryo is a useful method to treat infertility that shows embryo potential for selecting the best one to transfer and successfully implantation. However, embryo development in vitro is affected by oxidative stresses such as reactive oxygen species that may damage embryo development. Antioxidants are molecules found in fruits, vegetables, and fish that play an important role in reducing oxidative processes. In the natural environment, there is a physiological antioxidant system that protects embryos against oxidative damage. This antioxidant system does not exist in vitro. Antioxidants act as free radical scavengers and protect cells or repair damage done by free radicals. Various studies have shown that adding antioxidants into embryo culture medium improves embryo development in vitro. This review article emphasizes different aspects of various antioxidants, including types, functions and mechanisms, on the growth improvement of different species of embryos in vitro.

Pseudoplatystoma coruscans is a very popular species for tropical fish culture as it has boneless meat of delicate taste and firm texture. Few studies on fish reproductive biology refer to the morphological features of eggs. The goal, therefore, of this present work was to perform a structural and ultrastructural analysis of fertilization and embryonic development in P. coruscans. The incubation period, from fertilization to hatching, lasts 13 h at 28/29 °C and 18 h at 27 °C. The oocytes had a mean diameter of 0.95 mm and hatched larvae were 2.55 mm in diameter. Analysing their development, we observed round, yellow oocytes that bore a double chorion membrane and a single micropyle. At 10 s after fertilization, several spermatozoa were detected attached to the oocyte surface. After 1 min of development, a fertilization cone that obstructed the micropyle could be observed. Segmentation started between 20 and 30 min after fertilization, when the egg cell was then formed. The first cleavage occurred between 30 and 45 min after fertilization, prior to reaching the morula stage (75 and 90 min after fertilization). The epiboly movement started at 120 and 180 min after fertilization and ended at 360 and 480 min after fertilization. Differentiation between cephalic and caudal region was detected after 420 and 600 min after fertilization and larvae hatched between 780 and 1080 min after fertilization. Seven main embryonic development stages were identified: egg cell, cleavage, morula, blastula, gastrula, segmentation with differentiation between cephalic and caudal regions, and hatching.

This study aimed to compare the neonatal outcomes of transplanted fifth day (D5) or sixth day (D6) frozen–thawed blastocysts, aiming to provide guidance when choosing blastocyst recovery methods. The clinical data of 1109 patients that were performed for recovered blastocyst transplantation, as well as the data of 515 patients who had already delivered were analyzed retrospectively. The patients were divided into single- or twins-birth groups according to blastocyst formation time (D5 or D6) and the number of fetus(es). The implantation rate, clinical pregnancy rate and live birth rate of the D5 group were all significantly higher than those in the D6 group (P < 0.05), and the biochemical pregnancy rate in the D5 group was significantly lower than that in the D6 group (P = 0.049). Other factors, including birth weight, birth length, incidence of low birth weight and sex ratio, differed between the D5 and D6 groups, but the difference was not statistically significant (P > 0.05). In conclusion, the implantation rate, clinical pregnancy rate and live birth rate in the D5 group were all significantly higher than that in the D6 group. The birth weight at D6 was prone to be heavier no matter the birth group, and the sex ratio in the D5 group was higher than that at D6, the difference in neonatal outcomes between the two groups was not statistically significant.

Germinal-vesicle-stage oocytes enclosed with compact cumulus cell layers (COCs) were recovered from adult or prepubertal minke whale ovaries, and were vitrified in a solution containing 15% ethylene glycol, 15% DMSO and 0.5 M sucrose using either a Cryotop or an open-pulled straw (OPS) as the cryodevice. The post-warm COCs with normal morphology were cultured for 40 h in a 390 mosmol in vitro maturation medium, and oocytes extruding the first polar body were considered to be matured. The proportion of morphologically normal COCs after vitrification and warming was higher when the COCs were cryopreserved by Cryotop (adult origin, 88.4%; prepubertal origin, 80.8%) compared with the OPS (adult origin, 67.7%; prepubertal origin, 64.2%). The oocyte maturation rate was higher in the adult/Cryotop group (29.1%) compared with those of the prepubertal/Cryotop group (14.4%), the adult/OPS group (14.3%) and the prepubertal/OPS group (10.6%). These results indicate that the Cryotop is a better device than the OPS for vitrification of immature oocytes from adult minke whales.

The aim of the present study was to investigate the stability of six housekeeping genes, and the relative expression of growth factors (EGF, GDF-9, BMP-15, VEGF, FGF-2, BMP-6, IGF-1 and KL) and hormone receptors (FSH, LH and GH) in goat preantral follicles. To evaluate to stability of housekeeping genes micro-dissected fresh follicles (150–200 μm) as well as follicles that have been in vitro cultured for 12 days were used. In addition, isolated fresh follicles were used to compare expression of various growth factors and hormone receptors before culture. Both fresh and cultured follicles were subjected to total RNA extraction and synthesis of cDNA. After amplification of cDNA by real-time PCR, the geNorm software program was used to evaluate the stability of glyceraldehyde-2-phosphate dehydrogenase (GAPDH), β-tubulin, β-actin, phosphoglycerokinase (PGK), 18S rRNA, ubiquitin (UBQ) and ribosomal protein 19 (RPL-19). In addition, follicular steady-state levels of mRNA from the various growth factors under study were compared. Results demonstrated that, in goat preantral follicles, UBQ and β-actin were the most suitable reference genes and thus could be used as parameters to normalize data from future in vitro studies. In contrast, 18S RNA appeared the least stable gene among the tested housekeeping genes. Analysis of mRNA for several hypophyseal hormone receptors in fresh preantral follicles showed significantly higher FSH-R mRNA levels than those of LH-R and GH-R, and no difference between GH-R and LH-R mRNA levels. In regard growth factor mRNA expression in goat preantral follicles, EGF mRNA levels appeared significantly lower than those of the other studied growth factors. Increasingly higher relative mRNA levels were observed for GDF-9, BMP-15, BMP-6, FGF-2, VEGF, Kl and IGF-1, successively. In conclusion, UBQ and β-actin are the most stable housekeeping genes in fresh and 12-days cultured caprine preantral follicles. Furthermore, in fresh follicles, high levels of FSH-R mRNA are detected while among eight growth factors, IGF-1 is the most highly expressed and EGF the weakest expressed compound.

The objective of this study was to determine the effects of oviductal fluid and heparin on sperm penetration and the characteristics of spermatozoa. The addition of oviductal fluid and heparin to the fertilisation medium decreased sperm penetration and the mean number of spermatozoa in penetrated eggs. The number of spermatozoa firmly bound to zona pellucida was also decreased in the presence of oviductal fluid and heparin. Chlortetracycline (CTC) fluorescence patterns were used to determine the incidence of capacitation and the acrosome reaction. The proportion of capacitated and acrosome-free spermatozoa increased when spermatozoa were exposed for 1.5 and 3 h to oviductal fluid and heparin. In contrast heparin alone did not increase the number of capacitated spermatozoa at these time points. These results suggest that factor(s) in oviductal secretions reduce polyspermic fertilisation and the number of spermatozoa that will penetrate porcine oocytes. The reduction of polyspermic penetration by oviductal secretions may be due to a reduced number of spermatozoa in the fertilisation medium with an intact acrosome.

The cortical cytoplasm and cortical granules (CGs) of mouse oocytes were analysed by electron microscopy. Oocytes were collected soon and 20 h after ovulation from adult young females (3–4 months old). In addition, gametes collected soon after ovulation from 12- to 14-month-old females were used. Ultrastructural analyses were undertaken using the conventional procedures and the alcoholic PTA method. PTA selectively stains the CGs indicating the presence of lysine-rich proteins in these granules. Oocytes from young females showed CGs as dense granules 300–500 nm in diameter linearly arranged under the oolemma. In oocytes recovered 20 h after ovulation 24.31% of CGs appeared vacuolated and 38.40% internalized in the cytoplasm. In gametes collected from old females several changes were observed in the cortical cytoplasm: (a) CGs appeared concentrated in some areas while others regions were devoid of granules; (b) groups of CGs appeared internalized in the egg cytoplasm; (c) the CG contents had swollen and changed, showing dense and clear areas; (d) numerous dense structures and vesicles (lysosome-like vesicles) were present; (e) cytoplasmic fragmentation was frequently seen. Fragments contained CGs, dense structures and vacuoles. These changes are closely related to the low fertilization rates shown by these oocytes when they were used for in vitro fertilization procedures.

Xenopus laevis eggs are surrounded by an extracellular matrix consisting of a vitelline envelope, and three jelly layers, J1, J2, and J3 (from egg surface outward). The jelly layers vary in thickness (about 150, 15 and 200 μm for J1, J2 and J3 respectively) but all are translucent allowing observation of sperm penetration. Video microscopy demonstrated that sperm are able to penetrate and traverse J3 at velocities approaching 30 μm/s. Sperm swim through jelly in a corkscrew-like manner with their rotational and forward velocities being tightly coupled at about 30°/μm forward travel. They are propelled by whip-like power strokes involving hairpin bends in the flagellum that are generated every 180° of rotation and which are propagated from base to tip. The overall trajectories of individual sperm are quite variable. Many sperm head directly for J2 but some do not, these swimming circumferentially, or even away from the egg surface. Most sperm (over 97%) that enter the jelly do not get to the egg surface but are stopped at a variety of positions within J3 or at the outer surface of J2. Efficient sperm penetration and passage through the jelly layers requires a low electrolyte concentration in the surrounding medium, and is inhibited by the lectin wheat germ agglutin (WGA) in a dose-dependent manner. WGA does not block sperm penetration of J3 but does block further progression towards the egg surface. This observation suggests that sperm motility within the jelly is dependent on the carbohydrate moieties of the large glycoconjugates present, and that their alteration by WGA binding accounts for the inability of sperm to reach the egg surface and fertilise the egg.

We have used the whole-cell recording technique to compare three stages of primary and secondary oocytes from F1 hybrid mice (C57BL/6J x SJL/J):neonatal germinal vesicle (NGV) stage primary oocytes from 10- to 20-day-old, prepubescent mice; mature germinal vesicle (MGV) stage primary oocytes from 12-week-old, post-pubescent, superovulated mice; first polar body (FPB) stage secondary oocytes from 12-week-old, post-pubescent mice during the normal oestrus cycle or following superovulation. NGV, MGV and FPB oocytes all exhibit two voltage-dependent currents: an inward, rapidly activating/inactivating current, and an outward, slowly activating/non-inactivating current. In 1.5 mmol/1 external Ca the average peak inward current is − 2.9, − 12.4 and − 13.8 μA/cm2 in NGV, MGV and FPB oocytes, respectively. In 20 mmol/1 Ca these currents increase and the reversal potential shifts to the right. The outward current decreases slightly with growth and development: at 40 mV test potentials, NGV oocytes have average outward currents of 8.9 μA/cm2, and MGV and FPB oocytes have currents of 5.0 and 5.5 μA/cm2, respectively. Thus, MGV oocytes express FPB current patterns. The reversal potentials, kinetics and pharmacology of the currents indicate that Ca channels carry the inward current and K channels carry the outward current. During growth in vivo a gradual depolarisation accompanies maturation. Resting potentialsranged from − 45 to − 30 mV in NGV oocytes to − 35 to − 17 mV in MGV oocytes to − 20 mV to − 3 mV in FPB oocytes. These data suggest that a selective increase occurs in the number of Ca channels during oocyte growth. This increase precedes nuclear maturation and coincides with the acquisition of meiotic competence.

Intraspecific and interspecific cloning via somatic cell nuclear transfer (iSCNT) is a biotechnique with great possibilities for wild mammals because it allows the maintenance of biodiversity by recovering species, nuclear reprogramming for the production of pluripotency-induced cells, and studies related to embryonic development. Nevertheless, many areas in cloning, especially those associated with wild mammals, are still in question because of the difficulty in obtaining cytoplasmic donor cells (or cytoplasts). Conversely, donor cell nuclei (or karyoplasts) are widely obtained from the skin of living or post-mortem individuals and often maintained in somatic cell banks. Moreover, the creation of karyoplast–cytoplast complexes by fusion followed by activation and embryo development is one of the most difficult steps that requires further clarification to avoid genetic failures. Although difficult, cloning different species, such as wild carnivores and ungulates, can be successful via iSCNT with embryo development and the birth of offspring. Thus, novel research in the area that contributes to the conservation of biodiversity and knowledge of the physiology of species continues. The present review presents the failures and successes that occurred with the application of the technique in wild mammals, with the goal of helping future work on cloning via iSCNT.

The fertilisability and developmental capacity of mouse oocytes matured in vitro were examined by in vitro fertilisation (IVF) and intracytoplasmic sperm injection (ICSI). While more than 50% of cumulus-enclosed oocytes were fertilised by IVF after maturation in serum-supplemented medium, none were fertilised when the oocytes matured without serum. By ICSI, the majority (78-94%) of the oocytes were fertilised regardless of the presence or absence of serum in oocyte maturation media. Although the majority (88-92%) of cumulus-free germinal vesicle oocytes underwent nuclear maturation in both serum-free and serum-containing media, those matured in the presence of serum were more readily fertilised by ICSI (43%) than those matured without it (3-5%). The cumulus-free oocytes co-cultured with cumulus cells but without serum were fertilised at 36%, suggesting some secreted factor promotes the oocyte's cytoplasmic maturation. The oocytes fertilised by ICSI developed into normal-term fetuses regardless of the presence or absence of serum or cumulus cells in oocyte maturation medium. These results lead us to conclude that (a) the cytoplasm of the oocytes can mature in serum-free medium and (b) the presence of both the serum and the cumulus cells in the medium surrounding maturing oocytes is beneficial for the development of the fertilisation- and development-competence of oocyte cytoplasm.

Granulosa cells influence the growth and acquisition of the developmental competence of oocytes. We investigated the effects of ageing on the proliferative activity, global genomic DNA methylation, relative telomere length and telomerase activity of bovine granulosa cells. The proliferative activity of cells was examined by bromodeoxyuridine (BrdU) assay, genomic DNA methylation was examined by enzyme-linked immunosorbent assay (ELISA), and relative telomere length and telomerase activity were examined by real-time polymerase chain reaction. We first compared the proliferative activity of the granulosa cells of the medium follicles between in dominant phase ovaries and growth phase ovaries. We observed that the proliferative activity of the granulosa cells of dominant phase ovaries was significantly lower than those of growth phase ovaries. In addition, the proliferative activity of granulosa cells was inversely associated with follicular size. Based on the results, we used granulosa cells harvested from the medium follicles (3–5 mm in diameter) on the surfaces of the dominant phase ovaries collected from cows at a slaughterhouse. The proliferative activity of the granulosa cells harvested from the ovaries of old cows (N = 8; average age 165.1 months) was lower than that of the cells from young cows (N = 8; average age 30.9 months). Global loss of cytosine methylation was detected in the granulosa cells of old cows (N = 12; average age 141.0 months) compared with young cows (N = 15; average age 27.4 months). Although the relative telomere lengths of cumulus cells were similar in the two age groups, the relative telomere lengths and telomerase activity of the granulosa cells from old cows (N = 17 and 9; average age, 164.6 and 151.3 months, respectively) tended to be shorter than those of the cells from young cows (N = 17 and 10; average age 30.6 and 28.1 months, respectively); however, this difference was not significant p = 0.09 and 0.053, respectively). In conclusion, the proliferative activity and genomic global DNA methylation significantly decreased, and the relative telomere lengths and telomerase activity of granulosa cells tended to be shorter with the age of donor cows.

The African catfish Clarias gariepinus Burchell 1822 is a favourite aquaculture fish in many parts of Africa and Asia because of its hardiness and fast growth rate. In this study, early, post-embryonic and larval developmental stages of C. gariepinus were examined chronologically and described. Photomicrographs of unfertilized matured oocytes from 0 min of fertilization through all cell stages to alevin, to complete yolk absorption, to free swimming larval stages are shown and documented live from lateral and top views, with the aid of a light microscope. Extruded oocytes had a mean diameter of 1 ± 0.1 mm, and possessed a thin perivitelline membrane whose space was filled with a protoplasmic layer. Heartbeat was in the range of 115–160/min prior to hatching. Hatchability rate was 85% and hatching occurred at 17 h at a controlled temperature of 28.5 ± 0.5°C, while ontogeny of the eyes and other organs were discernible. At day 4, larvae mean length was 9.3 ± 0.5 mm, exogenous feeding had commenced fully and melanophores spread cephalocaudally but were concentrated significantly on the head parts. This paper, for the first time, presents the significant chronological developmental stages of C. gariepinus embryology that will have significant implications for genetic manipulation and catfish seed production for aquaculture.

The seminal plasma PSP-I/PSP-II spermadhesin is able to preserve, in vitro, the viability of highly extended boar spermatozoa, suggesting it might be used as a suitable ameliorator for the damaging effects of sperm handling, including in vitro fertilization. However, little is known about the ligand capability of PSP-I/PSP-II as regards the zona pellucida (ZP) or its possible role in gamete interaction. The present study evaluated the effect of the presence of PSP-I/PSP-II (1.5 mg/ml) during in vitro oocyte maturation and also during co-incubation of frozen-thawed boar spermatozoa with either immature (IM) or in vitro matured (IVM) oocytes, either enclosed by cumulus cells or denuded. Exposure of the gametes to the heterodimer during in vitro gamete co-incubation showed a significant blocking effect of sperm penetration rates and a decreased number of spermatozoa per oocyte in both IM and IVM denuded oocytes. Such an effect was not present in cumulus-enclosed oocytes, suggesting the effect could be mediated by exposed ZP receptors. In addition, when PSP-I/PSP-II was added to the IVM medium, oocyte maturation rates were significantly reduced. In conclusion, the results suggest that PSP-I/PSP-II, when present in vitro, blocks sperm–ZP binding.

Soybean trypsin inhibitor (SBTI) inhibits the catalytic activity of serine proteases, and has been shown to bind to acrosin, an acrosomal hydrolase which is not exposed on the surface of macaque sperm until after the acrosome reaction. Following activation with caffeine and dibutyryl cAMP, cynomolgus macaque sperm were induced to acrosome react with calcium ionophore A23187 in the presence of SBTI and were fixed for ultrastructural observation. Transmission electron microscopy (TEM) revealed secondary labelling of anti-SBTI-IgG with colloidal gold in association with the acrosomal matrix and fused membranes of sperm undergoing the acrosome reaction, but gold labelling was not observed on acrosome-intact sperm. When SBTI was conjugated with the fluorochrome Alexa 488, labelled (acrosome-reacted) sperm showed bright fluorescence that ranged from a patchy or punctate appearance to solid labelling over the region of the acrosomal cap. Following treatment with ionophore, the percentages of total acrosome-reacted sperm (motile and non-motile) as assessed with Alexa-SBTI, fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA), and TEM were 54.6%, 51.6% and 61.5%, respectively. Measures of acrosomal status with FITC-PSA and Alexa-SBTI were highly correlated (r = 0.94; n = 3). Macaque zonae pellucidae were co-incubated with activated sperm for 1 min and then rinsed in medium containing Alexa-SBTI and immediately observed with epifluorescence microscopy. The mean percentage of Alexa-SBTI-labelled (acrosome-reacted) motile sperm bound to the zona was 45.7 ± 14 (range: 22–80.4%; n = 4). Fewer than 1% of the motile sperm in suspension surrounding the zonae were acrosome-reacted. Alexa-SBTI had no effect on sperm motility, survival, or zona binding capability.

The miniature pig is regarded as a better organ donor breed for xenotransplantation than other pig breeds because the size of their organs is similar to that of humans. To improve efficiency of cloned miniature pig production, we analysed the effect of breed difference between donor cells and embryo recipients on pregnancy rate and delivery rate. Cloned porcine embryos derived from domestic or miniature pig donor cells were transferred to domestic or miniature recipient pigs. Delivery rate was significantly higher when embryos reconstructed with miniature pig donor cells were transferred to miniature pig recipients as compared with that of embryos transferred to domestic pig recipients. However, pregnancy rates were similar between the two groups. The breed of donor cells, but not of embryo recipients, seems likely to affect litter size. From a 13 610 gene cDNA microarray, 1551 (11.7%) genes showed significantly different levels of expression between the fetuses of the two breeds. Vascular endothelial growth factor and c-kit ligand genes related to implantation and maintenance of pregnancy were significantly down-regulated in miniature pigs. In conclusion, the differential gene expression in fetuses interferes with proper fetal/maternal interactions, and results in late-stage pregnancy loss. Our results indicate that the miniature pig is the preferred embryo recipient breed than domestic pig for producing cloned miniature piglets.

Experiments were undertaken to develop intracytoplasmic sperm injection (ICSI) to produce caprine embryos out of the normal breeding season. Oocytes were obtained from 2–6 mm ovarian follicles at slaughter. Selected oocytes with two to four layers of cumulus cells were incubated in 1 ml of H-TCM 199 supplemented with 10 μg each of oFSH and bLH (NHPP, NIDDK, NICHD, USDA) and 20% fetal bovine serum (FBS) in a thermos (38.5°C) for 4.5 h during transportation. Then, oocytes were transferred into 75 μl of freshly prepared maturation medium under paraffin oil and a mixture of 5% O2, 5% CO2 and 90% N2. Approximately 26 h after recovery oocytes were denuded by incubation with hyaluronidase (100 IU/ml) and pipetting and held at 38.5°C for 90 min. Spermatozoa frozen in egg yolk extender were thawed in a 37°C water bath for 15s. Motile fractions were selected by swim-up, then incubated for 90 mm in TALP with 10 μg heparin/ml. Each oocyte was positioned with its first polar body at 6 or 12 o'clock by a holding pipette. Sperm (1 μl) were added to 10 μl medium containing 10% polyvinylpyrrolidone. A sperm cell was aspirated into a pipette, and then injected head-first into the cytoplasm of an oocyte maintained in H-TCM 199 + 20% FBS at 37°C. Injected oocytes were transferred to HM and, after 90 min, cultured in 50 μl of BSA-free synthetic oviduct fluid plus polyvinyl alcohol, citrate and non-essential amino acids. Results demonstrate that caprine blastocysts can be produced outside the breeding season by the use of frozen-thawed semen and injection of sperm cells with broken tails into ova followed by culture in defined medium.

In assisted reproductive technology (ART) programmes, approximately 10% of infertile patients have at least two or three repeated implantation failures (RIFs) after an in vitro fertilization (IVF) protocol. Successful implantation mainly depends on local immune tolerance mechanisms involving a spectrum of cytokines, interleukins and growth factors. The latter have played pivotal roles in the recruitment of immune cells (and notably T-lymphocyte cells). In total, 250 couples participating in frozen–thawed embryo transfer programme were incorporated in a randomized clinical trial (peripheral blood mononuclear cells (PBMC) subgroup: n=122; control subgroup: n=128). In the PBMC group, a blood sample was collected 5 days before the scheduled frozen–thawed embryo transfer; PBMCs were isolated using Ficoll separation and then cultured for 72 h. Two days prior to embryo transfer, 0.4 ml of cultured PBMCs were transferred into the patient’s uterus. Although the clinical pregnancy rate was higher in the PBMC group (34.4%) than in the control group (23.4%), this difference was not statistically significant (P=0.05 in a chi-squared test). Nevertheless, when we limited the analysis to patients with ≥3 RIFs (n=138), there was a significant difference in the clinical pregnancy rate between the PBMC group (38.6%) and the control group (19.7%; P=0.01). Our results imply that PBMC transfer can be part of effective fertility treatment for patients with RIF.