Eye Infection with SARS-CoV-2 as a Route to Systemic Immunization?

Infectious diseases of the conjunctiva and cornea usually leave behind both broad local and systemic immunity. Case reports of SARS-CoV-2-positive conjunctivitis with subsequent systemic immunity suggest a new route of immunization preventing the primary infection of the airways. MATERIAL AND METHODS: A total of 24 Syrian field hamsters were treated. In systematic animal experiments, we infected the eyes of n = 8 animals (group 1) and the airways of another n = 8 animals (group 2) with SARS-CoV-2 (Wuhan type); n = 8 hamsters served as controls (group 3). The weight development of the animals was recorded. After two weeks of observation of disease symptoms, all animals were re-exposed to SARS-CoV-2 in the respiratory tract (challenge) to determine whether immunity to the virus had been achieved. RESULTS: The epi-ocularly infected animals (group 1) showed no clinically visible disease during the ocular infection phase. At most, there was a slightly reduced weight gain compared to the control group (group 3), while the respiratory infected animals (group 2) all lost weight, became lethargic, and slowly recovered after two weeks. After the challenge, none of the animals in groups 1 and 2 became ill again. The animals in the negative control (group 3) all became ill. Cytotoxic antibodies were detectable in the blood of the infected groups before and after challenge, with higher titers in the epi-ocularly infected animals. CONCLUSION: By epi-ocular infection with SARS-CoV-2, the development of systemic immunity with formation of cytotoxic antibodies without severe general disease could be observed in the experimental animals, which did not induce any more disease upon a second infection in the respiratory tract. Therefore, it can be concluded that a purely epi-ocular infection with SARS-CoV2 only induces a weak disease pattern followed by systemic immunity.

Relationship between follicular volume and oocyte competence, blastocyst development and live-birth rate: optimal follicle size for oocyte retrieval.

OBJECTIVE: To analyze oocyte competence in gonadotropin-releasing hormone agonist (GnRHa) stimulation cycles with regard to maturity, fertilization and blastocyst rate, as well as clinical outcome (pregnancy and live-birth rate), in relation to follicular volume, measured by three-dimensional transvaginal sonography (3D-TVS), and follicular fluid composition. METHODS: This was a prospective single-center study conducted between June 2012 and June 2014, including 118 ovum pick-ups with subsequent embryo transfer. Ovarian stimulation was performed using the GnRHa long protocol. Of 1493 follicles aspirated individually, follicular volume was evaluated successfully in 1236 using automated 3D-TVS during oocyte retrieval. Oocyte maturity and blastocyst development were tracked according to follicular volume. Intrafollicular concentrations of estradiol, testosterone, progesterone, luteinizing hormone, follicle-stimulating hormone and granulocyte-colony stimulating factor were quantified by immunoassay. Clinical outcome, in terms of implantation rate, (clinical) pregnancy rate, miscarriage and live-birth rate (LBR), was evaluated. RESULTS: Follicles were categorized, according to their volume, into three arbitrary groups, which included 196 small (8-12 mm/0.3-0.9 mL), 772 medium (13-23 mm/1-6 mL) and 268 large (≥ 24 mm/> 6 mL) follicles. Although oocyte recovery rate was significantly lower in small follicles compared with medium and large ones (63.8% vs 76.6% and 81.3%, respectively; P < 0.001), similar fertilization rates (85.1% vs 75.3% and 81.4%, respectively) and blastocyst rates (40.5% vs 40.6% and 37.2%, respectively) per mature metaphase II oocyte were observed. A trend towards higher LBR after transfer of blastocysts derived from small (< 1 mL) follicles compared with medium (1-6 mL) or large (> 6 mL) follicles (54.5% vs 42.0%, and 41.7%, respectively) was observed. No predictive value of follicular fluid biomarkers was identified. CONCLUSIONS: Our data indicate that the optimal follicular volume for a high yield of good quality blastocysts with good potential to lead to a live birth is 13-23 mm/1-6 mL. However, oocytes derived from small follicles (8-12 mm/0.3-0.9 mL) still have the capacity for normal development and subsequent delivery of healthy children, suggesting that aspiration of these follicles should be encouraged as this would increase the total number of blastocysts retrieved per stimulation. Copyright © 2017 ISUOG. Published by John Wiley & Sons Ltd.

Cryopreservation of chicken primordial germ cells by vitrification and slow freezing: A comparative study.

In the present study, we compare a classical slow freezing (SLF) method and an aseptic vitrification (Vitrif) technique to cryopreserve a stable primordial germ cell (PGCs) line issued from the Ardennaise chicken breed. Viability immediately after warming was close to 80% and did not differ between the two cryopreservation methods. Proliferation tended to be slower for both cryopreservation methods compared with controls, but the difference was significant only for Vitrif. No difference was found between the two methods after flow cytometry analysis of stage-specific embryonic antigen-1 expression and reverse transcription-polymerase chain reaction on several factors related to PGC phenotype. After 1 week in culture, all cryopreserved cells reached controls' main morphologic and expanding (viability/proliferation) features. However, SLF generated more unwanted cells clusters than Vitrif. After injection of the PGCs into recipient embryos, vitrified PGCs reported a clear, yet not significant, tendency to colonize the gonad at a higher rate than slow frozen PGCs. SLF in cryovials remains simple, inexpensive, and less technically demanding than Vitrif. Nevertheless, the intrinsic advantages of our aseptic Vitrif method and the present study suggest that this should be considered as safer than classical SLF for cryopreserving chicken PGCs.

Lower intracellular concentration of cryoprotectants after vitrification than after slow freezing despite exposure to higher concentration of cryoprotectant solutions.

STUDY QUESTION: What is the intracellular concentration of cryoprotectant (ICCP) in mouse zygotes during vitrification (VIT) and slow-freezing (SLF) cryopreservation procedures? SUMMARY ANSWER: Contrary to common beliefs, it was observed that the ICCP in vitrified zygotes is lower than after SLF, although the solutions used in VIT contain higher concentrations of cryoprotectants (CPs). WHAT IS KNOWN ALREADY: To reduce the likelihood of intracellular ice crystal formation, which has detrimental effects on cell organelles and membranes, VIT was introduced as an alternative to SLF to cryopreserve embryos and gametes. Combined with high cooling and warming rates, the use of high concentrations of CPs favours an intracellular environment that supports and maintains the transition from a liquid to a solid glass-like state devoid of crystals. Although the up-to-date publications are reassuring in terms of obstetric and perinatal outcomes after VIT, a fear about exposing gametes and embryos to high amounts of CPs that exceed 3-4-fold those found in SLF was central to a debate initiated by advocates of SLF procedures. STUDY DESIGN, SIZE, DURATION: Two experimental set-ups were applied. The objective of a first study was to determine the ICCP at the end of the exposure steps to the CP solutions with our VIT protocol (n = 31). The goal of the second investigation was to compare the ICCP between VIT (n = 30) and SLF (n = 30). All experiments were performed in triplicates using mouse zygotes. The study took place at the GIGA-Research Institute of the University of Liège. PARTICIPANTS/MATERIALS, SETTING, METHODS: Cell volume is modified by changes in extracellular osmolarity. Hence, we estimated the final ICCP after the incubation steps in the VIT solutions by exposing the cells to sucrose (SUC) solutions with defined molarities. The ICCP was calculated from the SUC concentration that produced no change in cell volume, i.e. when intra- and extracellular osmolarities were equivalent. Cell volume was monitored by microscopic cinematography. ICCP was compared between SLF and VIT based on the principle that a high ICCP lowers the probability of (re)crystallization during warming but increases the probability of over-swelling of the cell due to fast inflow of water. The survival rates of mouse zygotes after SLF or VIT were compared using either (i) various warming rates or (ii) various concentrations of SUC in the warming dilution medium. MAIN RESULTS AND THE ROLE OF CHANCE: The ICCP in mouse zygotes during the VIT procedure prior to plunging them in liquid nitrogen was ∼2.14 M, i.e. one-third of the concentration in the VIT solution. After SLF, the warming rate did not affect the zygote survival rate. In contrast, only 3/30 vitrified zygotes survived when warmed slowly but as many as 30/30 zygotes survived when warming was fast (>20 000°C/min). Vitrified zygotes showed significantly higher survival rates than slow-frozen zygotes when they were placed directly in the culture medium or in solutions containing low concentrations of SUC (P < 0.01). These two experiments demonstrate a lower ICCP after VIT than after SLF. LIMITATIONS, REASONS FOR CAUTION: The results should not be directly extrapolated to other stages of development or to other species due to possible differences in membrane permeability to water and CPs. WIDER IMPLICATIONS OF THE FINDINGS: The low ICCP we observed after VIT removes the concern about high ICCP after VIT, at least in murine zygotes and helps to explain the observed efficiency and lack of toxicity of VIT. STUDY FUNDING / COMPETING INTEREST(S): The study was funded by the FNRS (National Funds for Scientific Research). The authors declare that they have no competing interests.

[Closed carrier device: a reality to vitrify oocytes and embryos in aseptic conditions]. / Support fermé: une réalité clinique pour vitrifier en conditions aseptiques les ovocytes et embryons.

Vitrification with the use of "Open" carrier devices (Cryoloop, cryotop, cryoleaf, Vitriplug) which allowed the contact with liquid nitrogen has become a more popular way to achieve cooling rate superior to 20,000 °C/min. Even though the question of contamination with liquid nitrogen during ultra-rapid cooling and storage remain debatable with the use of "open" devices, it is important to revise the carrier system in a way, which minimizes the risk of contamination. According to the EU tissues and cells directive, it is advisable that the cooling and storage should be carried out in embryo carrier devices ensuring complete separation of the embryos from liquid nitrogen in a way, which minimizes the risk of contamination. The consequence of a reduction in the cooling rate resulting from the heat-insulating barrier of aseptic devices has to be counteracted by gradually increasing intracellular concentrations of cryoprotectants without inducing a toxic effect. We developed an aseptic vitrification method of vitrification for MII oocytes and embryos at different stage of development using the "VitriSafe" as "closed" carrier device.

[Vitrification and the use of high concentrations of cryoprotectants: is it a justified argument to prefer slow freezing?]. / Vitrification et utilisation de concentrations élevées en cryoprotecteurs: ceci justifie-t-il de préférer la congélation lente?

The use of high levels of cryoprotectants (CPs) in solutions applied to vitrify oocytes or embryos is an argument to still prefer slow freezing procedure. Is it a justified argument? Out of three studies using mice zygotes we may assume that (i) the intracellular concentration of CPs is far lower than the one in the vitrification solutions, (ii) the intracellular concentration of CPs in the vitrified zygote is in contrary to the common beliefs even lower than the one observed after a slow freezing procedure, (iii) survival after slow freezing reflects the presence of an intracellular vitrified state in these cells.

Higher sensitivity of Adamts12-deficient mice to tumor growth and angiogenesis.

ADAMTS (a disintegrin and metalloproteinase domain with thrombospondin motifs) constitute a family of endopeptidases related to matrix metalloproteinases. These proteases have been largely implicated in tissue remodeling and angiogenesis associated with physiological and pathological processes. To elucidate the in vivo functions of ADAMTS-12, we have generated a knockout mouse strain (Adamts12(-/-)) in which Adamts12 gene was deleted. The mutant mice had normal gestations and no apparent defects in growth, life span and fertility. By applying three different in vivo models of angiogenesis (malignant keratinocyte transplantation, Matrigel plug and aortic ring assays) to Adamts12(-/-) mice, we provide evidence for a protective effect of this host enzyme toward angiogenesis and cancer progression. In the absence of Adamts-12, both the angiogenic response and tumor invasion into host tissue were increased. Complementing results were obtained by using medium conditioned by cells overexpressing human ADAMTS-12, which inhibited vessel outgrowth in the aortic ring assay. This angioinhibitory effect of ADAMTS-12 was independent of its enzymatic activity as a mutated inactive form of the enzyme was similarly efficient in inhibiting endothelial cell sprouting in the aortic ring assay than the wild-type form. Altogether, our results show that ADAMTS-12 displays antiangiogenic properties and protect the host toward tumor progression.

Aseptic vitrification of blastocysts from infertile patients, egg donors and after IVM.

During embryo vitrification, it is advisable that cooling and storage should occur in a carrier device in which there is complete separation of the embryos from liquid nitrogen to ensure asepsis. The consequence of a reduction in the cooling rate resulting from the heat-insulating barrier aseptic devices has to be counteracted by gradually increasing intracellular concentrations of cryoprotectants without inducing a toxic effect. Blastocysts originating from couples with male and/or female factor infertility (group 1) or from oocyte donors (group 2) or from in-vitro matured oocytes (group 3) were gradually exposed to increasing concentrations of dimethylsulphoxide/ethylene glycol (5/5%, 10/10% and 20/20%) before aseptic vitrification using a specially designed carrier (VitriSafe), a modification of the open hemi-straw plug device. A total of 120 aseptic vitrification/warming cycles were performed in group 1, 91 in group 2 and 22 in group 3. Survival rates before embryo transfer, ongoing pregnancy and implantation rates were as follows: for group 1, 73, 43 and 26%; for group 2, 88, 53 and 34%; and for group 3, 69, 50 and 38%, respectively. In spite of reduced cooling rates due to aseptic vitrification conditions, a three-step exposure to cryoprotectant solutions protects the embryos effectively from cryo-injuries and guaranties high survival rates.

[Cryopreservation of human embryos by vitrification]. / Cryopréservation des embryons humains par vitrification.

Vitrification is a cryopreservation strategy where cells are converted into a glass-like amorphous solid which is free of any crystalline structure. Such process is achieved by a combination of high concentration of cryoprotectant and an extremely high cooling rate. In the last years, survival rates of up to 80% after thawing and pregnancy rates of almost 30% could be achieved after transfer of vitrified embryos at the zygote, cleavage, morula and blastocyst stages. Also deliveries of healthy babies have been reported numerous times. To this day, a limited interest in this technique can be noted. The explanation may lye in the apprehension of many ART units regarding exposure of embryos to high concentrations of cryoprotectants and storage in non sterile conditions. The aim of the first part of this article, is to analyse if such fears are justified on the basis that vitrification mimics conditions already in use for many years in slow-cooling procedures where cells are plunged into liquid nitrogen at around -30 degrees C and secondly since storage of embryos are now possible in high aseptic conditions. In the second part, results on survival after thawing, pregnancy rates and baby take home rates of vitrified embryos will be presented and the problems associated with vitrification of blastocysts will be discussed.

From the germinal cells to the newborn animal: the transmission of genes and life through the generations.

The technology of reproduction progressed considerably during the last decade, leading to a certain availability of in vitro methods for fertilisation, oocyte maturation and embryo culture. The most spectacular manipulations are cloning and transgenesis. This review focuses on the early appearance of germinal cell precursors and the long-standing fate of gametes in mammals. The evident complexity and long-term programming of events in gametes and early embryos explain part of the difficulties encountered during the development of in vitro and in vivo methods such as multiple ovulation and embryo transfer (MOET), oestrus synchronisation, ovulation induction, superovulation, in vitro maturation and fertilisation, cryopreservation, transgenesis, nuclear transfer and cloning) and the occurrence of unexpected alterations of development, e.g. embryonic or fetal mortality, large-weight newborn syndrome and other dysregulations in imprinting or DNA transmission.

Relationship of human follicular diameter with oocyte fertilization and development after in-vitro fertilization or intracytoplasmic sperm injection.

The aim of this work was to evaluate the relationship between follicular size at the time of oocyte retrieval, and the subsequent oocyte competence to be fertilized and to develop in vitro. All the obtained oocytes were classified according to the corresponding volume of aspirated follicular fluid. Aspirated volume of follicular fluid <2 ml corresponded to a follicular diameter <16 mm and constituted the small size group. Volume of follicular fluid from 2 to 6 ml corresponded to a diameter from 16 to 23 mm and constituted the medium size group. The large size group contained follicles with diameter >23 mm and corresponded to an aspirated volume of follicular fluid of >6 ml. A progressive and significant increase in the rates of oocytes with a first polar body was observed from the small size group to the other groups and from the medium to the large size group: 75.3, 85.9 and 95.3% respectively. After classical in-vitro fertilization (IVF), significantly better rates of fertilization and development were obtained in the medium size group compared to the two other groups. Moreover, a positive relationship was observed between follicular diameter and rates of embryos scored as 'good' when oocytes were fertilized by intracytoplasmic sperm injection (ICSI). These results demonstrated that follicular size is positively related to the oocyte ability to be fertilized and to develop. Although oocytes from small follicles gave lower percentages of development probably due to partial oocyte incompetence, they allowed an increase in the total number of embryos scored as 'good'.

Assessment of nuclear totipotency of fetal bovine diploid germ cells by nuclear transfer.

Nuclear transfer was used to study nuclear reprogramming of fetal diploid bovine germ cells collected at two stages of the fetal development. In the first case, germ cells of both sexes were collected during their period of intragonadal mitotic multiplication at 48 days post coïtum (d.p.c.). In the second case, only male germ cells were collected after this period, between 105 and 185 d.p.c. Isolated germ cells were fused with enucleated oocytes. Reconstituted embryos were cultured in vitro and those reaching the compacted morula or blastocyst stage were transferred into synchronous recipient heifers. Of 511 reconstituted embryos with 48 d.p.c. germ cells (309 males and 202 females), 48% (247/511 ) cleaved; 2.7% (14/511 ) reached the compacted morula stage and 8 of them the blastocyst stage (1.6%). No difference was observed between sexes. All 14 compacted morulae/blastocysts were transferred into 6 recipients and one pregnancy was initiated. This recipient was slaughtered at Day 35 and an abnormal conceptus (extended trophectoderm and degenerated embryo) was collected. Its male sex, genetically determined, corresponded to that of donor fetus. Of 380 reconstituted embryos with male 105 to 185 d.p.c. germ cells, 72.1% (274/380 ) cleaved, 2.1% (8 380 ) reached the compact morula stage and 7 of these the blastocyst stage (1.8%). Three blastocysts and one morula were transferred into 4 recipients. Two became pregnant at Day 21 but only one at Day 35 which aborted around Day 40. Our results show that the nucleus of diploid bovine germ cells of both sexes can be reprogrammed. However, in the absence of further development of these reconstituted embryos, nuclear totipotency of bovine diploid germ cells remains to be evidenced.

[Cloning using nuclear transfer in bovine species: results and perspectives]. / Le clonage par transfert de noyau dans l'espèce bovine: résultats et perspectives.

A all in vitro cloning technique was developed in which the reconstituted embryos from the first cycle nuclear transfer (cloning) were used as blastomere donor for the second cycle nuclear transfer (recloning). Such method permitted to produce 14.5% of morulae and 14.9% of blastocysts after the first and second cycles of nuclear transfer, respectively. The rates of birth obtained after transfer of such embryos were 21.4% et 20.8% for first and second cycles respectively, corresponding to 6 et 5 calves for 28 et 24 transferred embryos. Unfortunately, gestation pathologies and an increase of birth weights were observed. It seems that the in vitro presence of gametes and/or embryos may be responsible of an alteration in the control of gene expression.

[Genetic mechanism of human sex differentiation]. / Le déterminisme génétique de la différenciation sexuelle humaine.

The genetic mechanism controlling sexual differentiation had remained unknown for a long time. Karyotype analysis of sex-inverted patients or individuals with ambiguous sexual differentiation has enabled the localization and identification of genes involved. It is currently known that the SRY gene is responsible for the initiation of a cascade reaction leading to male differentiation of the primitive gonad. SRY is a +/- 820 base pairs gene located on the small arm of the Y chromosome, more precisely within the 1A1 alpha sub-segment. Although a few other genes are known to be involved in the downstream regulation of SRY, their precise mode of action is yet unknown.

Viability of cloned bovine embryos after one or two cycles of nuclear transfer and in vitro culture.

We described an exclusively in vitro procedure for cloning and recloning bovine embryos. Embryos obtained by IVM/IVF/IVC developed to the morula stage were used as blastomere donors in cunjunction with IVM recipient oocytes. Reconstructed embryos were developed in vitro in co-culture using bovine oviductal epithelial cells. The resulting morulae were used as donors for recloning under the same experimental conditions. No significant difference was observed between cloning and recloning in terms of development (rates of blastocysts: 12.9 versus 14.9%), in the number of nuclei per blastocyst (63.8 versus 49.1), or in pregnancy rates (35.7 versus 33.3%). The high variability observed between replicates and the correlation between results in first and second cycle nuclear transfer may suggest an inherant potential of individual donor embryos to support development by cloning.

Cytogenetic study of bovine oocytes matured in vitro.

Described in the present paper is a cytogenetic study of bovine oocytes matured in vitro. The cumulus-oocyte complexes (COC), punctured from ovaries recovered in a local slaughterhouse, were classified into 3 groups according to follicular diameter 1 to 4mm, 5 to 8mm and 9 to 13 mm. Metaphases available for observation were classified as metaphase I, haploid and diploid metaphase II. High levels of haploid metaphases II (90.6, 86.9 and 94.4 %) among the 3 groups of follicular sizes indicated successful meiotic resumption during in vitro maturation and suggested that cytoplasmic maturation may be responsible for low developmental rate after IVM, IVF and in vitro development (IVD).

Nuclear transplantation using bovine primordial germ cells from male fetuses.

The developmental potential of nuclei of bovine gonial cells was investigated by nuclear transfer. Gonial cells were collected from male fetuses at about 175 days post coitum (p.c.). They were fused with enucleated oocytes; reconstituted embryos were cultured in vitro for 7 days. Embryos reaching the compacted morula or blastocyst stage were either fixed for cell counting or transferred into recipients. Out of 115 oocyte-gonia fusions, 101 (87.8%) gave rise to cleaved embryos at Day 3 and 26 (22.6%) had reached the 8-cell stage. At Day 7, 1 (1%) developed to the morula stage and 5 (4%) reached the blastocyst stage. Three blastocysts were fixed and showed normal cell numbers (135; 90; 76 cells). Three blastocysts and one morula were transferred in four recipients; two recipients were pregnant at Day 21 but only one was positive at Day 35 p.c.; this last one aborted around Day 40 p.c. No conceptus was collected. These results indicate that gonial cell nuclei can be partially reprogrammed; they are able to develop into blastocysts and to initiate gestation. However, more experiments will be necessary to prove the nuclear totipotency of bovine gonial cells.

[Molecules of the family of aspartic proteinases in the placenta of ruminants: hormones or proteins?]. / Molécules de la famille des protéases aspartiques dans le placenta des ruminants: hormones ou protéines?

The ruminant placenta contains binucleate trophoblastic cells synthesizing proteins, migrating cross the barrier and fusing with endothelial cells of the endometrium. Recently described were two glycoproteins from the family of aspartic proteases, apparently lacking the enzymatic activity: the pregnancy associated glycoproteins I and II (PAGI and PAGII). The first (PAGI) is largely secreted in maternal blood, this characteristic copes with the lack of proteolytic activity. The second (PAGII) is not completely characterized. However, it binds to lutropin (LH) receptors with high affinity. This binding allows to assume that PAGII is likely the same as the bovine chorionic gonadotropin identified earlier (bCG). A better characterization of these glycoproteins (PAGI and PAGII) and other members of the family (PAGIII...) will answer these questions together with the unexplained invasive process of the placenta.

Preservation of oocyte and granulosa cell morphology in bovine preantral follicles cultured in vitro.

Described in the present paper is a culture system that preserves oocyte and granulosa cell morphology in bovine preantral follicles during 5 d in vitro. The effects of additional hypoxanthine and energy substrata (i.e., pyruvate and glutamine) on the morphology of cultured preantral follicles were investigated. It was shown that addition of a mixture of pyruvate, glutamine and hypoxantine to the culture medium increased the percentage of follicles with an intact oocyte from 29.4 to 78.6%. Morphological criteria are described to discriminate between normal and degenerated preantral follicles during culture by inverted microscopy. In addition, the importance of histological evaluation to judge the quality of oocyte and granulosa cells is demonstrated.

Development of a combined new mechanical and enzymatic method for the isolation of intact preantral follicles from fetal, calf and adult bovine ovaries.

The isolation of preantral follicles from the ovaries of bovine fetuses, calves and adult cows was performed using a simple, rapid mechanical and enzyme method. The ovaries were cut into small pieces with a tissue chopper. Then, the suspension was filtered successively through 500 and 100 mum nylon mesh filters. This simple mechanical procedure resulted in large numbers of isolated preantral follicles: 2,142 +/- 254; 512 +/- 92 and 298 +/- 54 from the ovaries of bovine fetuses, calves and cows, respectively. In addition, the ovarian fragments between 100 and 500 mum were suspended in 10 ml of M199 Hepes medium plus 5% FCS and divided into 2 equal parts: one portion was used for collagenase treatment (200 U/ml) for 20 minutes, while the other served as a control. Collagenase treatment resulted in 841 +/- 161; 216 +/- 51 and 52 +/- 17 preantral follicles from fetuses, calves and cows, respectively, compared with 312 +/- 86; 52 +/- 15 and 10 +/- 2 in the control group. The use of collagenase with ovarian fragments selected by filtration as a method for increasing the rate of recovery of preantral follicles is described here.